Methods for treating hematologic cancers

ABSTRACT

The present invention relates to methods of treating hematologic cancers using a combination of inhibitors of PD-1 or PD-L1 and TIM-3, LAG-3 or CTLA4.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/024,396, filed Mar. 24, 2016, now U.S. Pat. No. 10,570,204, which isa U.S. National Stage Application under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2014/057491, filed Sep. 25, 2014, which claims thebenefit of U.S. Provisional Application No. 61/882,702, filed Sep. 26,2013 and U.S. Provisional Application No. 62/017,192, filed Jun. 25,2014. The contents of the aforesaid applications are hereby incorporatedby reference in their entirety.

STATEMENT OF RIGHTS

This invention was made with government support under Grant NCATS8UL1TR000055 awarded by the National Institutes of Health. The U.S.government has certain rights in the invention. This statement isincluded solely to comply with 37 C.F.R. § 401.14(a)(f)(4) and shouldnot be taken as an assertion or admission that the application disclosesand/or claims only one invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 22, 2014, isnamed C2160-7004WO_SL.txt and is 46,210 bytes in size.

BACKGROUND OF THE INVENTION

Hematologic malignancies encompass cancers affecting blood, bone marrow,and lymph nodes and are particularly affected by modulation of theimmune system since immune system cells are derived from hematologiclineages. Although hematologic cancers have traditionally been treatedwith conventional drug therapies, such as alkylating and other DNAdamaging compounds, it is increasingly becoming recognized that immunecheckpoint regulators play critical roles in determining whetherhematologic cancer cells are tolerated or attacked by the immune system(Wu et al. (2012) Int. J. Biol. Sci. 8:1420-1430; Nirschl and Drake(2013) Clin. Cancer Res., electronically published July 18; Ceeraz etal. (2013) Trends Immunol., electronically published August 13).However, immune checkpoint regulators, such as CTLA-4, PD-1, VISTA,B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B,PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha(CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, LAG-3, BTLA,A2aR and many more, negatively regulate immune response progressionbased on complex and combinatorial interactions between numerous inputs.While some progress has been made to determine which interventions atwhich particular nodes of the immune checkpoint regulatory system can betargeted for benefiting the treatment of hematologic cancers (Kearl etal. (2013) J. Immunol. 190:5620-5628; Hallett et al. (2011) Biol. BloodMarrow Transplant. 17:1133-1145; Pardoll et al. (2012) Nat. Rev. Cancer12:252-264; Brahmer et al. (2012) N. Engl. J. Med. 366:2455-2465;Mocellin et al. (2013) Biochim. Biophys. Acta 1836:187-196; Topalian etal. (2012) N. Engl. J. Med. 366:2443-2454; and Wolchok et al. (2013) N.Engl. J. Med. 369:122-133), it is not currently possible to identifyspecific interactions having synergistic anti-cancer therapeuticefficacy. Accordingly, there is a great need in the art to definespecific and synergistic combinations of immune checkpoint regulatorsuseful for treating hematologic cancers.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery thatinhibiting or blocking Programmed Death 1 (PD-1) or Programmed Death-1Ligand (PD-L1) in combination with inhibition of an immune checkpointinhibitor (e.g., one or more of TIM-3, LAG-3 or CTLA4) results in asynergistic therapeutic benefit for treating a hematologic cancer, e.g.,a myeloma. This finding is unexpected given the lack of such benefitobserved for inhibiting or blocking other combinations of immunecheckpoint regulators.

Accordingly, in one aspect, the invention features a method of treatinga subject afflicted with a hematologic cancer comprising administeringto the subject an inhibitor of PD-1 or PD-L1, and an inhibitor of animmune checkpoint regulator (e.g., an inhibitor of one or more of TIM-3,LAG-3 or CTLA4). In one embodiment, an inhibitor of PD-1 or PD-L1 isadministered in combination with an inhibitor of TIM-3. In anotherembodiment, an inhibitor of PD-1 or PD-L1 is administered in combinationwith an inhibitor of LAG-3. In yet another embodiment, an inhibitor ofPD-1 or PD-L1 is administered in combination with an inhibitor ofCTLA-4. Inhibition as described herein can be performed by inhibition atthe DNA, RNA or protein level. In embodiments, an inhibitory nucleicacid (e.g., a dsRNA, siRNA or shRNA), can be used to inhibit expressionof an inhibitory molecule. In other embodiments, the inhibitor is apolypeptide, e.g., a soluble ligand, or an antibody or antigen-bindingfragment thereof, that binds to PD-1 or PD-L1, or other immunecheckpoint regulator. Examples of each of the aforesaid inhibitors areprovided in more detail below. The term “inhibition” or “inhibitor”includes a reduction in a certain parameter, e.g., an activity, of agiven molecule, e.g., an immune checkpoint regulator. For example,inhibition of an activity, e.g., a PD-1 activity, of at least 5%, 10%,20%, 30%, 40% or more is included by this term. Thus, inhibition neednot be 100%. Activities for the immune checkpoint regulators can bedetermined as described herein or assays known in the art.

In one embodiment, the inhibitor is a bispecific or multispecificantibody selective for PD-1 or PD-L1 and TIM-3, LAG-3 or CTLA4. Inanother embodiment, a combination of inhibitors comprising a firstinhibitor that selectively inhibits or blocks PD-1 or PD-L1 and a secondinhibitor that selectively inhibits or blocks TIM-3, LAG-3 or CTLA4 isprovided. In one embodiment, the inhibitor is a soluble ligand, e.g., asoluble ligand of PD-1, PD-L1, TIM-3, LAG-3 or CTLA-4 (e.g., aCTLA-4-Ig). In still another embodiment, the first inhibitor and/orsecond inhibitor is an antibody or an antigen binding fragment thereof,which specifically binds to PD-1 or PD-L1 and/or TIM-3, LAG-3 or CTLA4.In yet another embodiment, the antibody, or antigen binding fragmentthereof, is murine, chimeric, humanized, composite, or human. In anotherembodiment, the antibody, or antigen fragment thereof, is detectablylabeled, comprises an effector domain, comprises an Fc domain, and/or isselected from the group consisting of Fv, Fav, F(ab′)2), Fab′, dsFv,scFv, sc(Fv)2, and diabodies fragments. In still another embodiment, theantibody, or antigen binding fragment thereof, is conjugated to acytotoxic agent (e.g., a chemotherapeutic agent, a biologic agent, atoxin, a radioactive isotope, and the like).

For example, an anti-PD-1 or PD-L1 antibody, or antigen binding fragmentthereof, can be administered in combination with an anti-LAG-3 antibodyor an antigen-binding fragment thereof. In another embodiment, ananti-PD-1 or PD-L1 antibody, or antigen binding fragment thereof, isadministered in combination with an anti-TIM-3 antibody orantigen-binding fragment thereof. In yet other embodiments, an anti-PD-1or PD-L1 antibody, or antigen binding fragment thereof, is administeredin combination with an anti-LAG-3 antibody and an anti-TIM-3 antibody,or antigen-binding fragments thereof. In yet other embodiments, ananti-PD-1 or PD-L1 antibody, or antigen binding fragment thereof, isadministered in combination with an anti-CTLA-4 antibody, or antigenbinding fragment thereof (e.g., ipilimumab). Any combination of theaforesaid antibodies can be used in the methods described herein. Thecombination of antibodies recited herein can be administered separately,e.g., as separate antibodies, or linked, e.g., as a bispecific ortrispecific antibody molecule. In one embodiment, a bispecific antibodythat includes an anti-PD-1 or PD-L1 antibody, or antigen bindingfragment thereof, and an anti-TIM-3, anti-LAG-3 antibody or anti-CTLA4antibody, or antigen-binding fragment thereof, is administered.

In addition to, or in place of antibodies and antigen binding fragmentsthereof, numerous other agents are contemplated. For example, in oneembodiment, the agent comprises an RNA interfering agent which inhibitsexpression of PD-1 or PD-L1 and/or TIM-3, LAG-3 or CTLA4 (e.g., a smallinterfering RNA (siRNA), small hairpin RNA (shRNA), or a microRNA(miRNA)). In another embodiment, the agent comprises an antisenseoligonucleotide complementary to PD-1 or PD-L1 and/or TIM-3, LAG-3 orCTLA4. In still another embodiment, the agent comprises a peptide orpeptidomimetic that inhibits or blocks PD-1 or PD-L1 and/or TIM-3, LAG-3or CTLA4. In yet another embodiment, the agent comprises a smallmolecule that inhibits or blocks PD-1 or PD-L1 and/or TIM-3, LAG-3 orCTLA4 (e.g., a small molecule that inhibits a protein-proteininteraction between PD-L1 and a PD-L1 receptor and/or TIM-3 and a TIM-3receptor). In another embodiment, the agent comprises an aptamer thatinhibits or blocks PD-1 or PD-L1 and/or TIM-3, LAG-3 or CTLA4.

Numerous adaptations to the methods described herein are contemplated.For example, in one embodiment, that at least one agent is administeredin a pharmaceutically acceptable formulation. In another embodiment, themethod further comprises administering to the subject a therapeuticagent for treating the hematologic cancer. In still another embodiment,the method further comprises a step of transient or completelymphodepletion (e.g., sublethal whole body irradiation used fortransient lymphodepletion or lethal whole body irradiation used forcomplete lymphodepletion). In yet another embodiment, the step oflymphodepletion occurs before, concurrently with, or after the step ofagent administration. In another embodiment, the hematologic cancer isselected from the group consisting of multiple myeloma, acutelymphocytic leukemia, acute myeloid leukemia, chronic lymphocyticleukemia, small lymphocytic lymphoma, non-Hodgkin's lymphoma, Hodgkin'slymphoma, mantle cell lymphoma, follicular lymphoma, Waldenstrom'smacroglobulinemia, B-cell lymphoma and diffuse large B-cell lymphoma,precursor B-lymphoblastic leukemia/lymphoma, B-cell chronic lymphocyticleukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, splenic marginal zone B-cell lymphoma (withor without villous lymphocytes), hairy cell leukemia, plasma cellmyeloma/plasmacytoma, extranodal marginal zone B-cell lymphoma of theMALT type, nodal marginal zone B-cell lymphoma (with or withoutmonocytoid B cells), Burkitt's lymphoma; precursor T-lymphoblasticlymphoma/leukemia, T-cell prolymphocytic leukemia, T-cell granularlymphocytic leukemia, aggressive NK cell leukemia, adult T-celllymphoma/leukemia (HTLV 1-positive), nasal-type extranodal NK/T-celllymphoma, enteropathy-type T-cell lymphoma, hepatosplenic γ-δ T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosisfungoides/Sézary syndrome, anaplastic large cell lymphoma (T/null cell,primary cutaneous type), anaplastic large cell lymphoma (T-/null-cell,primary systemic type), peripheral T-cell lymphoma not otherwisecharacterized, angioimmunoblastic T-cell lymphoma, polycythemia vera(PV), myelodysplastic syndrome (MDS), indolent Non-Hodgkin's Lymphoma(iNHL) and aggressive Non-Hodgkin's Lymphoma (aNHL). In someembodiments, the hematologic cancer is selected from the groupconsisting of B-cell lymphoma, myeloid leukemia and multiple myeloma orcan be multiple myeloma alone. In another embodiment, the subject is amammal, optionally wherein the mammal is a human.

In another aspect, a kit for treating a subject afflicted with ahematologic cancer comprising an inhibitor of PD-1 or PD-L1, and aninhibitor of an immune checkpoint regulator (e.g., an inhibitor of oneor more of TIM-3, LAG-3 or CTLA4), is provided. In one embodiment, theinhibitor is a bispecific or multispecific antibody, or antigen bindingfragment thereof, selective for both PD-1 or PD-L1 and TIM-3, LAG-3 orCTLA4. Similarly, a kit for treating a subject afflicted with ahematologic cancer comprising a first agent that selectively inhibits orblocks PD-1 or PD-L1 and a second agent that selectively inhibits orblocks TIM-3, LAG-3 or CTLA4, is provided. In one embodiment, the firstagent and/or second agent is an antibody, or an antigen binding fragmentthereof, which specifically binds to PD-1 or PD-L1 protein and/or TIM-3,LAG-3 or CTLA4 protein. Any antibody, or antigen binding fragmentthereof, provided in a kit can be murine, chimeric, humanized,composite, or human. In another embodiment, the antibody, or antigenbinding fragment thereof, is detectably labeled, comprises an effectordomain, comprises an Fc domain, and/or is selected from the groupconsisting of Fv, Fav, F(ab′)2), Fab′, dsFv, scFv, sc(Fv)2, anddiabodies fragments. In still another embodiment, the antibody, orantigen binding fragment thereof, is conjugated to a cytotoxic agent(e.g., a chemotherapeutic agent, a biologic agent, a toxin, and aradioactive isotope). In yet another embodiment, the agent is selectedfrom the group consisting of a) an RNA interfering agent which inhibitsexpression of PD-1 or PD-L1 and/or TIM-3, LAG-3 or CTLA4, optionallywherein said RNA interfering agent is an small interfering RNA (siRNA),small hairpin RNA (shRNA), or a microRNA (miRNA); b) an antisenseoligonucleotide complementary to PD-1 or PD-L1 and/or TIM-3, LAG-3 orCTLA4; c) a peptide or peptidomimetics that inhibits or blocks PD-1 orPD-L1 and/or TIM-3, LAG-3 or CTLA4; d) a small molecule that inhibits orblocks PD-1 or PD-L1 and/or TIM-3, LAG-3 or CTLA4, optionally whereinsaid small molecule inhibits a protein-protein interaction between PD-L1and a PD-L1 receptor and/or other immune checkpoint regulator (e.g.,TIM-3 and a TIM-3 receptor); and e) an aptamer that inhibits or blocksPD-1 or PD-L1 and/or TIM-3, LAG-3 or CTLA4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows representative flow cytometry dot plots depictingaccumulation of tumor cells in myeloma bearing mice over time.

FIG. 1B shows percentages of CD4⁺ and CD8⁺ T cells expressing indicatedimmune checkpoint proteins in myeloma bearing mice over time. Data shownare representative of more than four independent analyses. *p<0.05,**p<0.01, ***p<0.001 as compared to T cells from naïve non-myelomabearing mice.

FIG. 1C shows expression of Tim-3 and PD-1, Lag-3 and PD-1, or 2B4 andPD-1 on gated CD8⁺ or CD4⁺ T cells from bone marrow of moribund mice.

FIG. 2 shows representative flow cytometry dot plots depictingexpression of indicated immune checkpoint proteins on CD4⁺ Treg cells inmyeloma bearing mice and control mice.

FIG. 3A shows expression of Tim-3 and PD-1, Lag-3 and PD-1, or 2B4 andPD-1 on gated CD8⁺ T cells from myeloma bearing mice treated withsublethal whole body irradiation and anti-PD-L1 antibody. Data shown arerepresentative of more than four independent analyses.

FIG. 3B shows frequency of CD8⁺Tim-3⁺, CD8⁺Lag-3⁺ and CD8⁺2B4⁺ cells inspleens of anti-PD-L1 antibody treated myeloma bearing mice comparedwith spleens of control antibody treated. ***p<0.001.

FIG. 4 shows a schematic diagram of the experimental treatment protocolused to establish and treat myelomas in mice in Example 1.

FIG. 5 shows survival data of myeloma bearing mice treated with interalia a blocking anti-PD-L1 antibody, a blocking anti-TIM-3 antibody, orcombinations thereof.

FIG. 6 shows survival data of myeloma bearing mice treated with interalia a blocking anti-PD-L1 antibody, a blocking anti-LAG-3 antibody, orcombinations thereof.

FIG. 7 shows survival data of myeloma bearing mice treated with interalia a blocking anti-TIM-3 antibody, a blocking anti-LAG-3 antibody, orcombinations thereof.

FIG. 8 shows survival data of myeloma bearing mice treated with interalia a blocking anti-PD-L1 antibody and a blocking anti-PD-1 antibody.

FIG. 9A shows a schematic diagram of the experimental treatment protocolused to establish and treat myelomas in mice in Example 2.

FIG. 9B shows survival curves of myeloma bearing mice treated with ablocking anti-Tim-3 antibody only, a blocking anti-Lag-3 antibody only,or in combination with a blocking anti-PD-L1 antibody. Survival wascompared with control antibody treated mice or mice treated withanti-PD-L1 antibody only. A combination of anti-Lag-3 and anti-Tim-3antibodies was also tested. Survival curves represent combined data fromthree independent experiments; n=10-15 mice per experimental group.

FIG. 9C shows survival curves of myeloma bearing mice treated with ablocking anti-CTLA4 antibody only, or in combination with a blockinganti-PD-L1 antibody. Survival was compared with control antibody treatedmice or mice treated with anti-PD-L1 antibody only. Survival curvesrepresent combined data from three independent experiments; n=10-15 miceper experimental group.

FIG. 9D shows survival curves of myeloma bearing mice treated with ablocking anti-CD48 antibody only, or in combination with a blockinganti-PD-L1 antibody. Survival was compared with control antibody treatedmice or mice treated with anti-PD-L1 antibody only. Survival curvesrepresent combined data from two independent experiments; n=10-15 miceper experimental group.

FIG. 9E shows survival curves of some of the survivors from FIGS. 9B-9Cre-challenged with 5T33 myeloma cells on day 110. P values weredetermined by the log-rank test.

FIG. 10A shows frequencies of tumor-reactive CD8⁺ and CD4⁺ T cells inthe spleens and bone marrow of mice treated with combinations of immunecheckpoint protein blockade. The graphs are representative of threeindependent experiments in which the CD8⁺ or CD4⁺ T cells for each groupwere pooled from 5-7 individual mice.

FIG. 10B shows levels of cytokine production by CD8⁺ T cells purifiedfrom the spleens of mice treated with combinations of immune checkpointprotein blockade. The graphs are representative of two independentexperiments in which the CD8⁺ T cells for each group were pooled from 5individual mice. *p<0.05, **p<0.01 as compared with T cells from micetreated with anti-PD-L1 alone.

FIG. 11A shows expression of PD-1 on gated CD8⁺ T cells from spleen andbone marrow of mice treated with different blocking antibodies orcontrol IgG.

FIG. 11B shows tumor-reactive IFN-γ-secreting cell frequencies in thepresence of anti-PD-L1 or control IgG (10 μg/ml). The graphs arerepresentative of two independent experiments in which the CD8⁺ T cellsfor each group were pooled from five to seven individual mice. P valueswere determined by the Student t test. *p<0.05, **p<0.01, ***p<0.001.

FIG. 12 shows levels of cytokine production by CD4⁺ T cells isolatedfrom spleens of myeloma bearing mice treated with combinations of immunecheckpoint protein blockade. The graphs are representative of twoindependent experiments in which the CD4⁺ T cells for each group werepooled from 5 individual mice. *p<0.05, **p<0.01 as compared with Tcells from mice treated with anti-PD-L1 alone.

FIG. 13 shows expression of immune checkpoint proteins PD-1, Tim-3,Lag-3 and 2B4 on CD4⁺ and CD8⁺ T cells in mice with other hematologiccancers.

FIG. 14 shows a working model of combined checkpoint blockade andlymphodepleting whole body irradiation.

DETAILED DESCRIPTION OF THE INVENTION

Methods are provided for treating a hematologic cancer, for example, invitro, ex vivo, or in vivo in a subject, comprising contacting acancerous cell or administering to a subject a therapeutically effectiveamount of at least one agent that selectively inhibits or blocks PD-1 orPD-L1 and TIM-3, LAG-3 or CTLA4. In some embodiments, the methodsinvolve a combination of an inhibitor of PD-1 or PD-L1 and an inhibitorof an immune checkpoint inhibitor (e.g., an inhibitor of one or more ofTIM-3, LAG-3 or CTLA4). In one embodiment, an inhibitor of PD-1 or PD-L1is administered in combination with an inhibitor of TIM-3. In anotherembodiment, an inhibitor of PD-1 or PD-L1 is administered in combinationwith an inhibitor of LAG-3. In yet another embodiment, an inhibitor ofPD-1 or PD-L1 is administered in combination with an inhibitor ofCTLA-4. Exemplary inhibitors include a bispecific antibody,multispecific antibody, or combination of individual antibodies thatinhibit or block an activity of PD-1 or PD-L1 and another immunecheckpoint inhibitor. Such combinations can provide therapeutic benefitfor treating hematologic cancers. Such discoveries are especiallysurprising and unexpected given reports regarding the lack of activitiesreported for immune checkpoint regulators, such as TIM-3, in human Tcell activation (see, for example, Leitner et al. (2013) PLoS Pathog.9:e1003253).

It will be appreciated that the methods and compositions describedherein may be combined with other treatment regimens and/or otherpredictive biomarkers and methods of using same. It will also beappreciated that the present invention is not limited to the particularembodiments described herein, but can be carried out in variations wellknown to the skilled artisan.

I. Definitions

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The “amount” of a marker, e.g., expression or copy number of a marker,or protein level of a marker, in a subject is “significantly” higher orlower than the normal amount of a marker, if the amount of the marker isgreater or less, respectively, than the normal level by an amountgreater than the standard error of the assay employed to assess amount,and preferably at least twice, and more preferably three, four, five,ten or more times that amount. Alternately, the amount of the marker inthe subject can be considered “significantly” higher or lower than thenormal amount if the amount is at least about two, and preferably atleast about three, four, or five times, higher or lower, respectively,than the normal amount of the marker.

The term “altered level of expression” of a marker refers to anexpression level or copy number of a marker in a test sample e.g., asample derived from a subject suffering from cancer, that is greater orless than the standard error of the assay employed to assess expressionor copy number, and is preferably at least twice, and more preferablythree, four, five or ten or more times the expression level or copynumber of the marker or chromosomal region in a control sample (e.g.,sample from a healthy subject not having the associated disease) andpreferably, the average expression level or copy number of the marker orchromosomal region in several control samples. The altered level ofexpression is greater or less than the standard error of the assayemployed to assess expression or copy number, and is preferably at leasttwice, and more preferably three, four, five or ten or more times theexpression level or copy number of the marker in a control sample (e.g.,sample from a healthy subject not having the associated disease) andpreferably, the average expression level or copy number of the marker inseveral control samples.

The term “altered activity” of a marker refers to an activity of amarker which is increased or decreased in a disease state, e.g., in acancer sample, as compared to the activity of the marker in a normal,control sample. Altered activity of a marker may be the result of, forexample, altered expression of the marker, altered protein level of themarker, altered structure of the marker, or, e.g., an alteredinteraction with other proteins involved in the same or differentpathway as the marker, or altered interaction with transcriptionalactivators or inhibitors.

Unless otherwise specified herein, the terms “antibody” and “antibodies”broadly encompass naturally-occurring forms of antibodies (e.g. IgG,IgA, IgM, IgE) and recombinant antibodies such as single-chainantibodies, chimeric and humanized antibodies and multi-specificantibodies, as well as fragments and derivatives of all of theforegoing, which fragments and derivatives have at least an antigenicbinding site. Antibody derivatives may comprise a protein or chemicalmoiety conjugated to an antibody.

The term “antibody” as used herein also includes an “antigen-bindingportion” of an antibody (or simply “antibody portion”). The term“antigen-binding portion”, as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., polypeptide or fragment thereof of PD-1, PD-L1, LAG-3,CTLA-4 and/or TIM-3). It has been shown that the antigen-bindingfunction of an antibody can be performed by fragments of a full-lengthantibody. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentpolypeptides (known as single chain Fv (scFv); see e.g., Bird et al.(1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16:778). Such single chain antibodies are also intended to be encompassedwithin the term “antigen-binding portion” of an antibody. Any VH and VLsequences of specific scFv can be linked to human immunoglobulinconstant region cDNA or genomic sequences, in order to generateexpression vectors encoding complete IgG polypeptides or other isotypes.VH and VL can also be used in the generation of Fab, Fv or otherfragments of immunoglobulins using either protein chemistry orrecombinant DNA technology. Other forms of single chain antibodies, suchas diabodies are also encompassed. Diabodies are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc.Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994)Structure 2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may bepart of larger immunoadhesion polypeptides, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionpolypeptides include use of the streptavidin core region to make atetrameric scFv polypeptide (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv polypeptides (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionpolypeptides can be obtained using standard recombinant DNA techniques,as described herein.

Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, orsyngeneic; or modified forms thereof (e.g., humanized, chimeric, etc.).Antibodies may also be fully human. Preferably, antibodies of theinvention bind specifically or substantially specifically to PD-1,PD-L1, LAG-3, CTLA-4 and/or TIM-3 polypeptides or fragments thereof.They may also be selective for such antigens such that they candistinguish such antigens from closely related antigens, such as otherB7 family members. The terms “monoclonal antibodies” and “monoclonalantibody composition”, as used herein, refer to a population of antibodypolypeptides that contain only one species of an antigen binding sitecapable of immunoreacting with a particular epitope of an antigen,whereas the term “polyclonal antibodies” and “polyclonal antibodycomposition” refer to a population of antibody polypeptides that containmultiple species of antigen binding sites capable of interacting with aparticular antigen. A monoclonal antibody composition typically displaysa single binding affinity for a particular antigen with which itimmunoreacts.

As used herein, a “blocking” antibody or an antibody “antagonist” or“inhibitor” is one which inhibits or reduces at least one biologicalactivity of the antigen(s) it binds. For example, an anti-PD-L1 oranti-TIM-3 antibody binds PD-L1 or TIM-3, respectively, and inhibits theability of PD-L1 to, for example, bind PD-1, and inhibits the ability ofTIM-3 to, for example, bind galectin-9 or phosphatidylserine. In certainembodiments, the blocking antibodies or antagonist antibodies orfragments thereof described herein substantially or completely inhibit agiven biological activity of the antigen(s).

The term “body fluid” refers to fluids that are excreted or secretedfrom the body as well as fluid that are normally not (e.g. amnioticfluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid,cerumen and earwax, cowper's fluid or pre-ejaculatory fluid, chyle,chyme, stool, female ejaculate, interstitial fluid, intracellular fluid,lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum,semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication,vitreous humor, vomit).

The term “bispecific antibody” or “multispecific antibody” refers to anantibody that recognized more than one epitope. Such antibodies areuseful for targeting different proteins using the same agent. Methods ofmaking such antibodies are well known in art (see, at least U.S. Pat.Nos. 5,798,229; 5,989,830; and Holliger et al. (2005) Nat. Biotech.23:1126-1136).

The terms “cancer” or “tumor” or “hyperproliferative disorder” refer tothe presence of cells possessing characteristics typical ofcancer-causing cells, such as uncontrolled proliferation, immortality,metastatic potential, rapid growth and proliferation rate, and certaincharacteristic morphological features. Cancer cells are often in theform of a tumor, but such cells may exist alone within an animal, or maybe a non-tumorigenic cancer cell, such as a leukemia cell. Cancersinclude, but are not limited to, B cell cancer, e.g., multiple myeloma,Waldenstrom's macroglobulinemia, the heavy chain diseases, such as, forexample, alpha chain disease, gamma chain disease, and mu chain disease,benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas,breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostatecancer, pancreatic cancer, stomach cancer, ovarian cancer, urinarybladder cancer, brain or central nervous system cancer, peripheralnervous system cancer, esophageal cancer, cervical cancer, uterine orendometrial cancer, cancer of the oral cavity or pharynx, liver cancer,kidney cancer, testicular cancer, biliary tract cancer, small bowel orappendix cancer, salivary gland cancer, thyroid gland cancer, adrenalgland cancer, osteosarcoma, chondrosarcoma, cancer of hematologictissues, and the like. Other non-limiting examples of types of cancersapplicable to the methods encompassed by the present invention includehuman sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, liver cancer,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, bone cancer, brain tumor, testicular cancer, lung carcinoma,small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g.,acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic,promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronicleukemia (chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, and heavy chain disease. In some embodiments, cancersare epithlelial in nature and include but are not limited to, bladdercancer, breast cancer, cervical cancer, colon cancer, gynecologiccancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, headand neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, orskin cancer. In other embodiments, the cancer is breast cancer, prostatecancer, lung cancer, or colon cancer. In still other embodiments, theepithelial cancer is non-small-cell lung cancer, nonpapillary renal cellcarcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovariancarcinoma), or breast carcinoma. The epithelial cancers may becharacterized in various other ways including, but not limited to,serous, endometrioid, mucinous, clear cell, Brenner, orundifferentiated.

The term “control” refers to any reference standard suitable to providea comparison to the expression products in the test sample. In oneembodiment, the control comprises obtaining a “control sample” fromwhich expression product levels are detected and compared to theexpression product levels from the test sample. Such a control samplemay comprise any suitable sample, including but not limited to a samplefrom a control cancer patient (can be stored sample or previous samplemeasurement) with a known outcome; normal tissue or cells isolated froma subject, such as a normal patient or the cancer patient, culturedprimary cells/tissues isolated from a subject such as a normal subjector the cancer patient, adjacent normal cells/tissues obtained from thesame organ or body location of the cancer patient, a tissue or cellsample isolated from a normal subject, or a primary cells/tissuesobtained from a depository. In another preferred embodiment, the controlmay comprise a reference standard expression product level from anysuitable source, including but not limited to housekeeping genes, anexpression product level range from normal tissue (or other previouslyanalyzed control sample), a previously determined expression productlevel range within a test sample from a group of patients, or a set ofpatients with a certain outcome (for example, survival for one, two,three, four years, etc.) or receiving a certain treatment (for example,standard of care cancer therapy). It will be understood by those ofskill in the art that such control samples and reference standardexpression product levels can be used in combination as controls in themethods of the present invention. In one embodiment, the control maycomprise normal or non-cancerous cell/tissue sample. In anotherpreferred embodiment, the control may comprise an expression level for aset of patients, such as a set of cancer patients, or for a set ofcancer patients receiving a certain treatment, or for a set of patientswith one outcome versus another outcome. In the former case, thespecific expression product level of each patient can be assigned to apercentile level of expression, or expressed as either higher or lowerthan the mean or average of the reference standard expression level. Inanother preferred embodiment, the control may comprise normal cells,cells from patients treated with combination chemotherapy, and cellsfrom patients having benign cancer. In another embodiment, the controlmay also comprise a measured value for example, average level ofexpression of a particular gene in a population compared to the level ofexpression of a housekeeping gene in the same population. Such apopulation may comprise normal subjects, cancer patients who have notundergone any treatment (i.e., treatment naive), cancer patientsundergoing standard of care therapy, or patients having benign cancer.In another preferred embodiment, the control comprises a ratiotransformation of expression product levels, including but not limitedto determining a ratio of expression product levels of two genes in thetest sample and comparing it to any suitable ratio of the same two genesin a reference standard; determining expression product levels of thetwo or more genes in the test sample and determining a difference inexpression product levels in any suitable control; and determiningexpression product levels of the two or more genes in the test sample,normalizing their expression to expression of housekeeping genes in thetest sample, and comparing to any suitable control. In particularlypreferred embodiments, the control comprises a control sample which isof the same lineage and/or type as the test sample. In anotherembodiment, the control may comprise expression product levels groupedas percentiles within or based on a set of patient samples, such as allpatients with cancer. In one embodiment a control expression productlevel is established wherein higher or lower levels of expressionproduct relative to, for instance, a particular percentile, are used asthe basis for predicting outcome. In another preferred embodiment, acontrol expression product level is established using expression productlevels from cancer control patients with a known outcome, and theexpression product levels from the test sample are compared to thecontrol expression product level as the basis for predicting outcome. Asdemonstrated by the data below, the methods of the invention are notlimited to use of a specific cut-point in comparing the level ofexpression product in the test sample to the control.

The term “hematologic cancer” refers to cancers of cells derived fromthe blood. In some embodiments, the hematologic cancer is selected fromthe group consisting of acute lymphocytic leukemia, myeloid leukemiaincluding acute myeloid leukemia and chronic myelogenous leukemia,chronic lymphocytic leukemia, small lymphocytic lymphoma, multiplemyeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, mantle celllymphoma, follicular lymphoma, Waldenstrom's macroglobulinemia, B-celllymphoma and including diffuse large B-cell lymphoma (including primarymediastinal B-cell lymphoma and intravascular large B-cell lymphoma),follicular lymphoma, chronic lymphocytic leukemia, small lymphocyticlymphoma, mantle cell lymphoma, mucosa-associated lymphoid tissue (MALT)lymphomas 9 e.g., extranodal marginal zone B-cell lymphoma of the MALTtype, nodal marginal zone B-cell lymphoma (with or without monocytoid Bcells)), marginal zone B-cell lymphomas (e.g., nodal marginal zoneB-cell lymphoma and splenic marginal zone B-cell lymphoma (with orwithout villous lymphocytes)), Burkitt lymphoma, lymphoplasmacyticlymphoma (Waldenstrom macroglobulinemia), mediastinal large B celllymphoma, precursor B-lymphoblastic leukemia/lymphoma and, B-cellchronic lymphocytic leukemia/small lymphocytic lymphoma, B-cellprolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginalzone B-cell lymphoma (with or without villous lymphocytes), hairy cellleukemia, plasma cell myeloma/plasmacytoma, extranodal marginal zoneB-cell lymphoma of the MALT type, nodal marginal zone B-cell lymphoma(with or without monocytoid B cells), Burkitt's lymphoma; precursorT-lymphoblastic lymphoma/leukemia, T-cell prolymphocytic leukemia,T-cell granular lymphocytic leukemia, aggressive NK cell leukemia, adultT-cell lymphoma/leukemia (HTLV 1-positive), nasal-type extranodalNK/T-cell lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic γ-δT-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosisfungoides/Sézary syndrome, anaplastic large cell lymphoma (T/null cell,primary cutaneous type), anaplastic large cell lymphoma (T-/null-cell,primary systemic type), peripheral T-cell lymphoma not otherwisecharacterized, angioimmunoblastic T-cell lymphoma, polycythemia vera(PV), myelodysplastic syndrome (MDS). NHL may include indolentNon-Hodgkin's Lymphoma (iNHL) or aggressive Non-Hodgkin's Lymphoma(aNHL).

As used herein, the term “coding region” refers to regions of anucleotide sequence comprising codons which are translated into aminoacid residues, whereas the term “noncoding region” refers to regions ofa nucleotide sequence that are not translated into amino acids (e.g., 5′and 3′ untranslated regions).

As used herein, the term “complementary” refers to the broad concept ofsequence complementarity between regions of two nucleic acid strands orbetween two regions of the same nucleic acid strand. It is known that anadenine residue of a first nucleic acid region is capable of formingspecific hydrogen bonds (“base pairing”) with a residue of a secondnucleic acid region which is antiparallel to the first region if theresidue is thymine or uracil. Similarly, it is known that a cytosineresidue of a first nucleic acid strand is capable of base pairing with aresidue of a second nucleic acid strand which is antiparallel to thefirst strand if the residue is guanine. A first region of a nucleic acidis complementary to a second region of the same or a different nucleicacid if, when the two regions are arranged in an antiparallel fashion,at least one nucleotide residue of the first region is capable of basepairing with a residue of the second region. Preferably, the firstregion comprises a first portion and the second region comprises asecond portion, whereby, when the first and second portions are arrangedin an antiparallel fashion, at least about 50%, and preferably at leastabout 75%, at least about 90%, or at least about 95% of the nucleotideresidues of the first portion are capable of base pairing withnucleotide residues in the second portion. More preferably, allnucleotide residues of the first portion are capable of base pairingwith nucleotide residues in the second portion.

As used herein, the term “determining a suitable treatment regimen forthe subject” is taken to mean the determination of a treatment regimen(i.e., a single therapy or a combination of different therapies that areused for the prevention and/or treatment of the cancer in the subject)for a subject that is started, modified and/or ended based oressentially based or at least partially based on the results of theanalysis according to the present invention. One example is starting anadjuvant therapy after surgery whose purpose is to decrease the risk ofrecurrence, another would be to modify the dosage of a particularchemotherapy. The determination can, in addition to the results of theanalysis according to the present invention, be based on personalcharacteristics of the subject to be treated. In most cases, the actualdetermination of the suitable treatment regimen for the subject will beperformed by the attending physician or doctor.

“Homologous” as used herein, refers to nucleotide sequence similaritybetween two regions of the same nucleic acid strand or between regionsof two different nucleic acid strands. When a nucleotide residueposition in both regions is occupied by the same nucleotide residue,then the regions are homologous at that position. A first region ishomologous to a second region if at least one nucleotide residueposition of each region is occupied by the same residue. Homologybetween two regions is expressed in terms of the proportion ofnucleotide residue positions of the two regions that are occupied by thesame nucleotide residue. By way of example, a region having thenucleotide sequence 5′-ATTGCC-3′ and a region having the nucleotidesequence 5′-TATGGC-3′ share 50% homology. Preferably, the first regioncomprises a first portion and the second region comprises a secondportion, whereby, at least about 50%, and preferably at least about 75%,at least about 90%, or at least about 95% of the nucleotide residuepositions of each of the portions are occupied by the same nucleotideresidue. More preferably, all nucleotide residue positions of each ofthe portions are occupied by the same nucleotide residue.

As used herein, the term “host cell” is intended to refer to a cell intowhich a nucleic acid of the invention, such as a recombinant expressionvector of the invention, has been introduced. The terms “host cell” and“recombinant host cell” are used interchangeably herein. It should beunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

The term “humanized antibody,” as used herein, is intended to includeantibodies made by a non-human cell having variable and constant regionswhich have been altered to more closely resemble antibodies that wouldbe made by a human cell. For example, by altering the non-human antibodyamino acid sequence to incorporate amino acids found in human germlineimmunoglobulin sequences. Humanized antibodies may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo), for example in the CDRs. The term“humanized antibody”, as used herein, also includes antibodies in whichCDR sequences derived from the germline of another mammalian species,such as a mouse, have been grafted onto human framework sequences.

As used herein, the term “immune cell” refers to cells that play a rolein the immune response. Immune cells are of hematopoietic origin, andinclude lymphocytes, such as B cells and T cells; natural killer cells;myeloid cells, such as monocytes, macrophages, eosinophils, mast cells,basophils, and granulocytes.

As used herein, the term “immune checkpoints” or “immune checkpointregulators” means a group of molecules on the cell surface of CD4⁺ andCD8⁺ T cells. These molecules fine-tune immune responses bydown-modulating or inhibiting an immune response, e.g., an anti-tumorimmune response. Immune checkpoint proteins are known in the art andinclude, without limitation, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1,B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR familyreceptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4(CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, LAG-3, BTLA, and A2aR (see,for example, WO 2012/177624). Immunotherapeutic agents that can act asimmune checkpoint inhibitors useful in the methods of the presentinvention, include, but are not limited to, inhibitors of PD-1, PD-L1,TIM-3, LAG-3 and CTLA-4 (e.g., soluble peptide inhibitors or antibodies,e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4antibodies, anti-TIM-3 antibodies, and anti-LAG-3 antibodies).

As used herein, the term “immune response” includes T cell mediatedand/or B cell mediated immune responses. Exemplary immune responsesinclude T cell responses, e.g., cytokine production and cellularcytotoxicity. In addition, the term immune response includes immuneresponses that are indirectly effected by T cell activation, e.g.,antibody production (humoral responses) and activation of cytokineresponsive cells, e.g., macrophages.

As used herein, the term “immunotherapeutic agent” can include anymolecule, peptide, antibody or other agent which can stimulate a hostimmune system to generate an immune response to a tumor or cancer in thesubject. Various immunotherapeutic agents are useful in the compositionsand methods described herein.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

As used herein, the term “inhibit” refers to any decrease in, forexample a particular action, function, or interaction. For example,cancer is “inhibited” if at least one symptom of the cancer is reduced,slowed, or delayed. As used herein, cancer is also “inhibited” ifrecurrence or metastasis of the cancer is reduced, slowed, or delayed.

As used herein, the term “interaction,” when referring to an interactionbetween two molecules, refers to the physical contact (e.g., binding) ofthe molecules with one another. Generally, such an interaction resultsin an activity (which produces a biological effect) of one or both ofsaid molecules.

An “isolated antibody” is intended to refer to an antibody that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds PD-L1polypeptide or a fragment thereof, or TIM-3 polypeptide or a fragmentthereof, is substantially free of antibodies that specifically bindantigens other than said polypeptide or a fragment thereof). Moreover,an isolated antibody may be substantially free of other cellularmaterial and/or chemicals.

As used herein, an “isolated protein” refers to a protein that issubstantially free of other proteins, cellular material, separationmedium, and culture medium when isolated from cells or produced byrecombinant DNA techniques, or chemical precursors or other chemicalswhen chemically synthesized. An “isolated” or “purified” protein orbiologically active portion thereof is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue sourcefrom which the antibody, polypeptide, peptide or fusion protein isderived, or substantially free from chemical precursors or otherchemicals when chemically synthesized. The language “substantially freeof cellular material” includes preparations, in which compositions ofthe invention are separated from cellular components of the cells fromwhich they are isolated or recombinantly produced. In one embodiment,the language “substantially free of cellular material” includespreparations of having less than about 30%, 20%, 10%, or 5% (by dryweight) of cellular material. When an antibody, polypeptide, peptide orfusion protein or fragment thereof, e.g., a biologically active fragmentthereof, is recombinantly produced, it is also preferably substantiallyfree of culture medium, i.e., culture medium represents less than about20%, more preferably less than about 10%, and most preferably less thanabout 5% of the volume of the protein preparation.

A “kit” is any manufacture (e.g. a package or container) comprising atleast one reagent, e.g. a probe or small molecule, for specificallydetecting and/or affecting the expression of a marker of the invention.The kit may be promoted, distributed, or sold as a unit for performingthe methods of the present invention. The kit may comprise one or morereagents necessary to express a composition useful in the methods of thepresent invention. In certain embodiments, the kit may further comprisea reference standard, e.g., a nucleic acid encoding a protein that doesnot affect or regulate signaling pathways controlling cell growth,division, migration, survival or apoptosis. One skilled in the art canenvision many such control proteins, including, but not limited to,common molecular tags (e.g., green fluorescent protein andbeta-galactosidase), proteins not classified in any of pathwayencompassing cell growth, division, migration, survival or apoptosis byGeneOntology reference, or ubiquitous housekeeping proteins. Reagents inthe kit may be provided in individual containers or as mixtures of twoor more reagents in a single container. In addition, instructionalmaterials which describe the use of the compositions within the kit canbe included.

A “marker” is a gene whose altered level of expression in a tissue orcell from its expression level in normal or healthy tissue or cell isassociated with a disease state, such as cancer. A “marker nucleic acid”is a nucleic acid (e.g., mRNA, cDNA) encoded by or corresponding to amarker of the invention. Such marker nucleic acids include DNA (e.g.,cDNA) comprising the entire or a partial sequence of any of the nucleicacid sequences set forth in the Sequence Listing or the complement ofsuch a sequence. The marker nucleic acids also include RNA comprisingthe entire or a partial sequence of any of the nucleic acid sequencesset forth in the Sequence Listing or the complement of such a sequence,wherein all thymidine residues are replaced with uridine residues. A“marker protein” is a protein encoded by or corresponding to a marker ofthe invention. A marker protein comprises the entire or a partialsequence of any of the sequences set forth in the Sequence Listing. Theterms “protein” and “polypeptide” are used interchangeably.

The “normal” level of expression of a marker is the level of expressionof the marker in cells of a subject, e.g., a human patient, notafflicted with a cancer. An “over-expression” or “significantly higherlevel of expression” of a marker refers to an expression level in a testsample that is greater than the standard error of the assay employed toassess expression, and is preferably at least twice, and more preferably2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 times or more higher than the expression activity or level of themarker in a control sample (e.g., sample from a healthy subject nothaving the marker associated disease) and preferably, the averageexpression level of the marker in several control samples. A“significantly lower level of expression” of a marker refers to anexpression level in a test sample that is at least twice, and morepreferably 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 times or more lower than the expression level of themarker in a control sample (e.g., sample from a healthy subject nothaving the marker associated disease) and preferably, the averageexpression level of the marker in several control samples.

An “over-expression” or “significantly higher level of expression” of amarker refers to an expression level in a test sample that is greaterthan the standard error of the assay employed to assess expression, andis preferably at least twice, and more preferably 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5,9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or morehigher than the expression activity or level of the marker in a controlsample (e.g., sample from a healthy subject not having the markerassociated disease) and preferably, the average expression level of themarker in several control samples. A “significantly lower level ofexpression” of a marker refers to an expression level in a test samplethat is at least twice, and more preferably 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or morelower than the expression level of the marker in a control sample (e.g.,sample from a healthy subject not having the marker associated disease)and preferably, the average expression level of the marker in severalcontrol samples.

The term “peripheral blood cell subtypes” refers to cell types normallyfound in the peripheral blood including, but is not limited to,eosinophils, neutrophils, T cells, monocytes, NK cells, granulocytes,and B cells.

The term “probe” refers to any molecule which is capable of selectivelybinding to a specifically intended target molecule, for example, anucleotide transcript or protein encoded by or corresponding to amarker. Probes can be either synthesized by one skilled in the art, orderived from appropriate biological preparations. For purposes ofdetection of the target molecule, probes may be specifically designed tobe labeled, as described herein. Examples of molecules that can beutilized as probes include, but are not limited to, RNA, DNA, proteins,antibodies, and organic molecules.

The term “prognosis” includes a prediction of the probable course andoutcome of cancer or the likelihood of recovery from the disease. Insome embodiments, the use of statistical algorithms provides a prognosisof cancer in an individual. For example, the prognosis can be surgery,development of a clinical subtype of cancer (e.g., hematologic cancers,such as multiple myeloma), development of one or more clinical factors,development of intestinal cancer, or recovery from the disease.

The term “response to cancer therapy” or “outcome of cancer therapy”relates to any response of the hyperproliferative disorder (e.g.,cancer) to a cancer therapy, preferably to a change in tumor mass and/orvolume after initiation of neoadjuvant or adjuvant chemotherapy.Hyperproliferative disorder response may be assessed, for example forefficacy or in a neoadjuvant or adjuvant situation, where the size of atumor after systemic intervention can be compared to the initial sizeand dimensions as measured by CT, PET, mammogram, ultrasound orpalpation. Response may also be assessed by caliper measurement orpathological examination of the tumor after biopsy or surgical resectionfor solid cancers. Responses may be recorded in a quantitative fashionlike percentage change in tumor volume or in a qualitative fashion like“pathological complete response” (pCR), “clinical complete remission”(cCR), “clinical partial remission” (cPR), “clinical stable disease”(cSD), “clinical progressive disease” (cPD) or other qualitativecriteria. Assessment of hyperproliferative disorder response may be doneearly after the onset of neoadjuvant or adjuvant therapy, e.g., after afew hours, days, weeks or preferably after a few months. A typicalendpoint for response assessment is upon termination of neoadjuvantchemotherapy or upon surgical removal of residual tumor cells and/or thetumor bed. This is typically three months after initiation ofneoadjuvant therapy. In some embodiments, clinical efficacy of thetherapeutic treatments described herein may be determined by measuringthe clinical benefit rate (CBR). The clinical benefit rate is measuredby determining the sum of the percentage of patients who are in completeremission (CR), the number of patients who are in partial remission (PR)and the number of patients having stable disease (SD) at a time point atleast 6 months out from the end of therapy. The shorthand for thisformula is CBR=CR+PR+SD over 6 months. In some embodiments, the CBR fora particular cancer therapeutic regimen is at least 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. In otherembodiments, the percentage of patients who are in either CR, PR, and/orSD in any combination at least 30 days, 2 months, 3 months, 4 months, 5months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12months, 18 months, 24 months, 30 months, 36 months, 60 months, or longeris at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, or more. In some embodiments, the percentage is 100% over such atime period. Additional criteria for evaluating the response to cancertherapies are related to “survival,” which includes all of thefollowing: survival until mortality, also known as overall survival(wherein said mortality may be either irrespective of cause or tumorrelated); “recurrence-free survival” (wherein the term recurrence shallinclude both localized and distant recurrence); metastasis freesurvival; disease free survival (wherein the term disease shall includecancer and diseases associated therewith). The length of said survivalmay be calculated by reference to a defined start point (e.g., time ofdiagnosis or start of treatment) and end point (e.g., death, recurrenceor metastasis). In addition, criteria for efficacy of treatment can beexpanded to include response to chemotherapy, probability of survival,probability of metastasis within a given time period, and probability oftumor recurrence. For example, in order to determine appropriatethreshold values, a particular cancer therapeutic regimen can beadministered to a population of subjects and the outcome can becorrelated to copy number, level of expression, level of activity, etc.of a marker determined prior to administration of any cancer therapy.The outcome measurement may be pathologic response to therapy given inthe neoadjuvant setting. Alternatively, outcome measures, such asoverall survival and disease-free survival can be monitored over aperiod of time for subjects following cancer therapy for whom themeasurement values are known. In certain embodiments, the same doses ofcancer therapeutic agents are administered to each subject. In relatedembodiments, the doses administered are standard doses known in the artfor cancer therapeutic agents. The period of time for which subjects aremonitored can vary. For example, subjects may be monitored for at least2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60months. Biomarker threshold values that correlate to outcome of a cancertherapy can be determined using methods such as those described in theExamples section. Outcomes can also be measured in terms of a “hazardratio” (the ratio of death rates for one patient group to another;provides likelihood of death at a certain time point), “overallsurvival” (OS), and/or “progression free survival.” In certainembodiments, the prognosis comprises likelihood of overall survival rateat 1 year, 2 years, 3 years, 4 years, or any other suitable time point.The significance associated with the prognosis of poor outcome in allaspects of the present invention is measured by techniques known in theart. For example, significance may be measured with calculation of oddsratio. In a further embodiment, the significance is measured by apercentage. In one embodiment, a significant risk of poor outcome ismeasured as odds ratio of 0.8 or less or at least about 1.2, includingby not limited to: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 4.0, 5.0, 10.0, 15.0, 20.0,25.0, 30.0 and 40.0. In a further embodiment, a significant increase orreduction in risk is at least about 20%, including but not limited toabout 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% and 98%. In a further embodiment, a significant increase inrisk is at least about 50%. Thus, the present invention further providesmethods for making a treatment decision for a cancer patient, comprisingcarrying out the methods for prognosing a cancer patient according tothe different aspects and embodiments of the present invention, and thenweighing the results in light of other known clinical and pathologicalrisk factors, in determining a course of treatment for the cancerpatient. For example, a cancer patient that is shown by the methods ofthe invention to have an increased risk of poor outcome by combinationchemotherapy treatment can be treated with more aggressive therapies,including but not limited to radiation therapy, peripheral blood stemcell transplant, bone marrow transplant, or novel or experimentaltherapies under clinical investigation.

The term “resistance” refers to an acquired or natural resistance of acancer sample or a mammal to a cancer therapy (i.e., being nonresponsiveto or having reduced or limited response to the therapeutic treatment),such as having a reduced response to a therapeutic treatment by 25% ormore, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2-fold,3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more. The reductionin response can be measured by comparing with the same cancer sample ormammal before the resistance is acquired, or by comparing with adifferent cancer sample or a mammal who is known to have no resistanceto the therapeutic treatment. A typical acquired resistance tochemotherapy is called “multidrug resistance.” The multidrug resistancecan be mediated by P-glycoprotein or can be mediated by othermechanisms, or it can occur when a mammal is infected with a multidrug-resistant microorganism or a combination of microorganisms. Thedetermination of resistance to a therapeutic treatment is routine in theart and within the skill of an ordinarily skilled clinician, forexample, can be measured by cell proliferative assays and cell deathassays as described herein as “sensitizing.” In some embodiments, theterm “reverses resistance” means that the use of a second agent incombination with a primary cancer therapy (e.g., chemotherapeutic orradiation therapy) is able to produce a significant decrease in tumorvolume at a level of statistical significance (e.g., p<0.05) whencompared to tumor volume of untreated tumor in the circumstance wherethe primary cancer therapy (e.g., chemotherapeutic or radiation therapy)alone is unable to produce a statistically significant decrease in tumorvolume compared to tumor volume of untreated tumor. This generallyapplies to tumor volume measurements made at a time when the untreatedtumor is growing log rhythmically.

An “RNA interfering agent” as used herein, is defined as any agent whichinterferes with or inhibits expression of a target gene, e.g., a markerof the invention, by RNA interference (RNAi). Such RNA interferingagents include, but are not limited to, nucleic acid molecules includingRNA molecules which are homologous to the target gene, e.g., a marker ofthe invention, or a fragment thereof, short interfering RNA (siRNA), andsmall molecules which interfere with or inhibit expression of a targetgene by RNA interference (RNAi).

“RNA interference (RNAi)” is an evolutionally conserved process wherebythe expression or introduction of RNA of a sequence that is identical orhighly similar to a target gene results in the sequence specificdegradation or specific post-transcriptional gene silencing (PTGS) ofmessenger RNA (mRNA) transcribed from that targeted gene (see Coburn andCullen (2002) J. Virol. 76(18):9225), thereby inhibiting expression ofthe target gene. In one embodiment, the RNA is double stranded RNA(dsRNA). This process has been described in plants, invertebrates, andmammalian cells. In nature, RNAi is initiated by the dsRNA-specificendonuclease Dicer, which promotes processive cleavage of long dsRNAinto double-stranded fragments termed siRNAs. siRNAs are incorporatedinto a protein complex that recognizes and cleaves target mRNAs. RNAican also be initiated by introducing nucleic acid molecules, e.g.,synthetic siRNAs or RNA interfering agents, to inhibit or silence theexpression of target genes. As used herein, “inhibition of target geneexpression” or “inhibition of marker gene expression” includes anydecrease in expression or protein activity or level of the target gene(e.g., a marker gene of the invention) or protein encoded by the targetgene, e.g., a marker protein of the invention. The decrease may be of atleast 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as comparedto the expression of a target gene or the activity or level of theprotein encoded by a target gene which has not been targeted by an RNAinterfering agent.

The term “sample” used for detecting or determining the presence orlevel of at least one biomarker is typically whole blood, plasma, serum,saliva, urine, stool (e.g., feces), tears, and any other bodily fluid(e.g., as described above under the definition of “body fluids”), or atissue sample (e.g., biopsy) such as a small intestine, colon sample, orsurgical resection tissue. In certain instances, the method of thepresent invention further comprises obtaining the sample from theindividual prior to detecting or determining the presence or level of atleast one marker in the sample.

The term “sensitize” means to alter cancer cells or tumor cells in a waythat allows for more effective treatment of the associated cancer with acancer therapy (e.g., chemotherapeutic or radiation therapy. In someembodiments, normal cells are not affected to an extent that causes thenormal cells to be unduly injured by the cancer therapy (e.g.,chemotherapy or radiation therapy). An increased sensitivity or areduced sensitivity to a therapeutic treatment is measured according toa known method in the art for the particular treatment and methodsdescribed herein below, including, but not limited to, cellproliferative assays (Tanigawa N, Kern D H, Kikasa Y, Morton D L, CancerRes 1982; 42: 2159-2164), cell death assays (Weisenthal L M, Shoemaker RH, Marsden J A, Dill P L, Baker J A, Moran E M, Cancer Res 1984; 94:161-173; Weisenthal L M, Lippman M E, Cancer Treat Rep 1985; 69:615-632; Weisenthal L M, In: Kaspers G J L, Pieters R, Twentyman P R,Weisenthal L M, Veerman A J P, eds. Drug Resistance in Leukemia andLymphoma. Langhorne, P A: Harwood Academic Publishers, 1993: 415-432;Weisenthal L M, Contrib Gynecol Obstet 1994; 19: 82-90). The sensitivityor resistance may also be measured in animal by measuring the tumor sizereduction over a period of time, for example, 6 month for human and 4-6weeks for mouse. A composition or a method sensitizes response to atherapeutic treatment if the increase in treatment sensitivity or thereduction in resistance is 25% or more, for example, 30%, 40%, 50%, 60%,70%, 80%, or more, to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold,20-fold or more, compared to treatment sensitivity or resistance in theabsence of such composition or method. The determination of sensitivityor resistance to a therapeutic treatment is routine in the art andwithin the skill of an ordinarily skilled clinician. It is to beunderstood that any method described herein for enhancing the efficacyof a cancer therapy can be equally applied to methods for sensitizinghyperproliferative or otherwise cancerous cells (e.g., resistant cells)to the cancer therapy.

“Short interfering RNA” (siRNA), also referred to herein as “smallinterfering RNA” is defined as an agent which functions to inhibitexpression of a target gene, e.g., by RNAi. An siRNA may be chemicallysynthesized, may be produced by in vitro transcription, or may beproduced within a host cell. In one embodiment, siRNA is a doublestranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides inlength, preferably about 15 to about 28 nucleotides, more preferablyabout 19 to about 25 nucleotides in length, and more preferably about19, 20, 21, or 22 nucleotides in length, and may contain a 3′ and/or 5′overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5nucleotides. The length of the overhang is independent between the twostrands, i.e., the length of the over hang on one strand is notdependent on the length of the overhang on the second strand. Preferablythe siRNA is capable of promoting RNA interference through degradationor specific post-transcriptional gene silencing (PTGS) of the targetmessenger RNA (mRNA). In another embodiment, an siRNA is a small hairpin(also called stem loop) RNA (shRNA). In one embodiment, these shRNAs arecomposed of a short (e.g., 19-25 nucleotide) antisense strand, followedby a 5-9 nucleotide loop, and the analogous sense strand. Alternatively,the sense strand may precede the nucleotide loop structure and theantisense strand may follow. These shRNAs may be contained in plasmids,retroviruses, and lentiviruses and expressed from, for example, the polIII U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003)RNA April; 9(4):493-501 incorporated by reference herein). RNAinterfering agents, e.g., siRNA molecules, may be administered to asubject having or at risk for having cancer, to inhibit expression of amarker gene of the invention, e.g., a marker gene which is overexpressedin cancer (such as the markers listed in Table 3) and thereby treat,prevent, or inhibit cancer in the subject.

As used herein, “subject” refers to any healthy animal, mammal or human,or any animal, mammal or human afflicted with a cancer, e.g., lung,ovarian, pancreatic, liver, breast, prostate, and colon carcinomas, aswell as melanoma and multiple myeloma. The term “subject” isinterchangeable with “subject”.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of antibody, polypeptide, peptide orfusion protein in which the protein is separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. In one embodiment, the language “substantially free of chemicalprecursors or other chemicals” includes preparations of antibody,polypeptide, peptide or fusion protein having less than about 30% (bydry weight) of chemical precursors or non-antibody, polypeptide, peptideor fusion protein chemicals, more preferably less than about 20%chemical precursors or non-antibody, polypeptide, peptide or fusionprotein chemicals, still more preferably less than about 10% chemicalprecursors or non-antibody, polypeptide, peptide or fusion proteinchemicals, and most preferably less than about 5% chemical precursors ornon-antibody, polypeptide, peptide or fusion protein chemicals.

As used herein, the term “survival” includes all of the following:survival until mortality, also known as overall survival (wherein saidmortality may be either irrespective of cause or tumor related);“recurrence-free survival” (wherein the term recurrence shall includeboth localized and distant recurrence); metastasis free survival;disease free survival (wherein the term disease shall include cancer anddiseases associated therewith). The length of said survival may becalculated by reference to a defined start point (e.g. time of diagnosisor start of treatment) and end point (e.g. death, recurrence ormetastasis). In addition, criteria for efficacy of treatment can beexpanded to include response to chemotherapy, probability of survival,probability of metastasis within a given time period, and probability oftumor recurrence.

The term “synergistic effect” refers to the combined effect of two ormore anticancer agents can be greater than the sum of the separateeffects of the anticancer agents or alone. In some embodiments, in canprovide for similar efficacy of monotherapy but with other unexpectedimprovements relative to monotherapy, such as reducing unwanted sideeffects.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living human cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

A “transcribed polynucleotide” or “nucleotide transcript” is apolynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA orcDNA) which is complementary to or homologous with all or a portion of amature mRNA made by transcription of a marker of the invention andnormal post-transcriptional processing (e.g. splicing), if any, of theRNA transcript, and reverse transcription of the RNA transcript.

An “underexpression” or “significantly lower level of expression or copynumber” of a marker refers to an expression level or copy number in atest sample that is greater than the standard error of the assayemployed to assess expression or copy number, but is preferably at leasttwice, and more preferably three, four, five or ten or more times lessthan the expression level or copy number of the marker in a controlsample (e.g., sample from a healthy subject not afflicted with cancer)and preferably, the average expression level or copy number of themarker in several control samples.

As used herein, the term “vector” refers to a nucleic acid capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” or simply “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

There is a known and definite correspondence between the amino acidsequence of a particular protein and the nucleotide sequences that cancode for the protein, as defined by the genetic code (shown below)Likewise, there is a known and definite correspondence between thenucleotide sequence of a particular nucleic acid and the amino acidsequence encoded by that nucleic acid, as defined by the genetic code.

GENETIC CODE Alanine (Ala, A) GCA, GCC, GCG, GCT Arginine (Arg, R) AGA,ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AAT Aspartic acid (Asp,D) GAC, GAT Cysteine (Cys, C) TGC, TGT Glutamic acid (Glu, E) GAA, GAGGlutamine (Gln, Q) CAA, CAG Glycine (Gly, G) GGA, GGC, GGG, GGTHistidine (His, H) CAC, CAT Isoleucine (Ile, I) ATA, ATC, ATT Leucine(Leu, L) CTA, CTC, CTG, CTT, TTA, TTG Lysine (Lys, K) AAA, AAGMethionine (Met, M) ATG Phenylalanine (Phe, F) TTC, TTT Proline (Pro, P)CCA, CCC, CCG, CCT Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCTThreonine (Thr, T) ACA, ACC, ACG, ACT Tryptophan (Trp, W) TGG Tyrosine(Tyr, Y) TAC, TAT Valine (Val, V) GTA, GTC, GTG, GTT Termination signal(end) TAA, TAG, TGA

An important and well known feature of the genetic code is itsredundancy, whereby, for most of the amino acids used to make proteins,more than one coding nucleotide triplet may be employed (illustratedabove). Therefore, a number of different nucleotide sequences may codefor a given amino acid sequence. Such nucleotide sequences areconsidered functionally equivalent since they result in the productionof the same amino acid sequence in all organisms (although certainorganisms may translate some sequences more efficiently than they doothers). Moreover, occasionally, a methylated variant of a purine orpyrimidine may be found in a given nucleotide sequence. Suchmethylations do not affect the coding relationship between thetrinucleotide codon and the corresponding amino acid.

In view of the foregoing, the nucleotide sequence of a DNA or RNA codingfor a fusion protein or polypeptide of the invention (or any portionthereof) can be used to derive the fusion protein or polypeptide aminoacid sequence, using the genetic code to translate the DNA or RNA intoan amino acid sequence. Likewise, for fusion protein or polypeptideamino acid sequence, corresponding nucleotide sequences that can encodethe fusion protein or polypeptide can be deduced from the genetic code(which, because of its redundancy, will produce multiple nucleic acidsequences for any given amino acid sequence). Thus, description and/ordisclosure herein of a nucleotide sequence which encodes a fusionprotein or polypeptide should be considered to also include descriptionand/or disclosure of the amino acid sequence encoded by the nucleotidesequence. Similarly, description and/or disclosure of a fusion proteinor polypeptide amino acid sequence herein should be considered to alsoinclude description and/or disclosure of all possible nucleotidesequences that can encode the amino acid sequence.

Finally, nucleic acid and amino acid sequence information for the lociand biomarkers of the present invention (e.g., biomarkers listed inTable 1) are well known in the art and readily available on publiclyavailable databases, such as the National Center for BiotechnologyInformation (NCBI). For example, exemplary nucleic acid and amino acidsequences derived from publicly available sequence databases areprovided below.

For example, the term “PD-1” refers to a member of the immunoglobulingene superfamily that functions as a coinhibitory receptor having PD-L1and PD-L2 as known ligands. PD-1 was previously identified using asubtraction cloning based approach to select for proteins involved inapoptotic cell death. PD-1 is a member of the CD28/CTLA-4 family ofmolecules based on its ability to bind to PD-L1. Like CTLA-4, PD-1 israpidly induced on the surface of T-cells in response to anti-CD3 (Agataet al. 25 (1996) Int. Immunol. 8:765). In contrast to CTLA-4, however,PD-1 is also induced on the surface of B-cells (in response toanti-IgM). PD-1 is also expressed on a subset of thymocytes and myeloidcells (Agata et al. (1996) supra; Nishimura et al. (1996) Int. Immunol.8:773).

The nucleic acid and amino acid sequences of a representative human PD-1biomarker is available to the public at the GenBank database under NM005018.2 and NP 005009.2 and is shown in Table 1 (see also Ishida et al.(1992) 20 EMBO J 11:3887; Shinohara et al. (1994) Genomics 23:704; U.S.Pat. No. 5,698,520). PD-1 has an extracellular region containingimmunoglobulin superfamily domain, a transmembrane domain, and anintracellular region including an immunoreceptor tyrosine-basedinhibitory motif (ITIM) (Ishida et al. (1992) EMBO J. 11:3887; Shinoharaet al. (1994) Genomics 23:704; and U.S. Pat. No. 5,698,520). Thesefeatures also define a larger family of polypeptides, called theimmunoinhibitory receptors, which also includes gp49B, PIR-B, and thekiller inhibitory receptors (KIRs) (Vivier and Daeron (1997) Immunol.Today 18:286). It is often assumed that the tyrosyl phosphorylated ITIMmotif of these receptors interacts with SH2-domain containingphosphatases, which leads to inhibitory signals. A subset of theseimmunoinhibitory receptors bind to MHC polypeptides, for example theKIRs, and CTLA4 binds to B7-1 and B7-2. It has been proposed that thereis a phylogenetic relationship between the MHC and B7 genes (Henry etal. (1999) Immunol. Today 20(6):285-8). Nucleic acid and polypeptidesequences of PD-1 orthologs in organisms other than humans are wellknown and include, for example, mouse PD-1 (NM_008798.2 andNP_032824.1), rat PD-1 (NM_001106927.1 and NP_001100397.1), dog PD-1(XM_543338.3 and XP_543338.3), cow PD-1 (NM_001083506.1 andNP_001076975.1), and chicken PD-1 (XM_422723.3 and XP_422723.2).

PD-1 polypeptides are inhibitory receptors capable of transmitting aninhibitory signal to an immune cell to thereby inhibit immune celleffector function, or are capable of promoting costimulation (e.g., bycompetitive inhibition) of immune cells, e.g., when present in soluble,monomeric form. Preferred PD-1 family members share sequence identitywith PD-1 and bind to one or more B7 family members, e.g., B7-1, B7-2,PD-1 ligand, and/or other polypeptides on antigen presenting cells.

The term “PD-1 activity,” includes the ability of a PD-1 polypeptide tomodulate an inhibitory signal in an activated immune cell, e.g., byengaging a natural PD-1 ligand on an antigen presenting cell. PD-1transmits an inhibitory signal to an immune cell in a manner similar toCTLA4. Modulation of an inhibitory signal in an immune cell results inmodulation of proliferation of, and/or cytokine secretion by, an immunecell. Thus, the term “PD-1 activity” includes the ability of a PD-1polypeptide to bind its natural ligand(s), the ability to modulateimmune cell costimulatory or inhibitory signals, and the ability tomodulate the immune response.

The term “PD-1 ligand” refers to binding partners of the PD-1 receptorand includes both PD-L1 (Freeman et al. (2000) J. Exp. Med. 192:1027)and PD-L2 (Latchman et al. (2001) Nat. Immunol. 2:261). At least twotypes of human PD-1 ligand polypeptides exist. PD-1 ligand proteinscomprise a signal sequence, and an IgV domain, an IgC domain, atransmembrane domain, and a short cytoplasmic tail. Both PD-L1 (SeeFreeman et al. (2000) J. Exp. Med. 192:1027 for sequence data) and PD-L2(See Latchman et al. (2001) Nat. Immunol. 2:261 for sequence data) aremembers of the B7 family of polypeptides. Both PD-L1 and PD-L2 areexpressed in placenta, spleen, lymph nodes, thymus, and heart. OnlyPD-L2 is expressed in pancreas, lung and liver, while only PD-L1 isexpressed in fetal liver. Both PD-1 ligands are upregulated on activatedmonocytes and dendritic cells, although PD-L1 expression is broader. Forexample, PD-L1 is known to be constitutively expressed and upregulatedto higher levels on murine hematopoietic cells (e.g., T cells, B cells,macrophages, dendritic cells (DCs), and bone marrow-derived mast cells)and non-hematopoietic cells (e.g., endothelial, epithelial, and musclecells), whereas PD-L2 is inducibly expressed on DCs, macrophages, andbone marrow-derived mast cells (see, Butte et al. (2007) Immunity27:111).

PD-1 ligands comprise a family of polypeptides having certain conservedstructural and functional features. The term “family” when used to referto proteins or nucleic acid molecules, is intended to mean two or moreproteins or nucleic acid molecules having a common structural domain ormotif and having sufficient amino acid or nucleotide sequence homology,as defined herein. Such family members can be naturally or non-naturallyoccurring and can be from either the same or different species. Forexample, a family can contain a first protein of human origin, as wellas other, distinct proteins of human origin or alternatively, cancontain homologues of non-human origin. Members of a family may alsohave common functional characteristics. PD-1 ligands are members of theB7 family of polypeptides. The term “B7 family” or “B7 polypeptides” asused herein includes costimulatory polypeptides that share sequencehomology with B7 polypeptides, e.g., with B7-1 (CD80), B7-2 (CD86),inducible costimulatory ligand (ICOS-L), B7-H3, B7-H4, VISTA, B7-H6, B7h(Swallow et al. (1999) Immunity 11:423), and/or PD-1 ligands (e.g.,PD-L1 or PD-L2). For example, human B7-1 and B7-2 share approximately26% amino acid sequence identity when compared using the BLAST programat NCBI with the default parameters (Blosum62 matrix with gap penaltiesset at existence 11 and extension 1 (see the NCBI website). The term B7family also includes variants of these polypeptides which are capable ofmodulating immune cell function. The B7 family of molecules share anumber of conserved regions, including signal domains, IgV domains andthe IgC domains. IgV domains and the IgC domains are art-recognized Igsuperfamily member domains. These domains correspond to structural unitsthat have distinct folding patterns called Ig folds. Ig folds arecomprised of a sandwich of two β sheets, each consisting ofanti-parallel β strands of 5-10 amino acids with a conserved disulfidebond between the two sheets in most, but not all, IgC domains of Ig,TCR, and MHC molecules share the same types of sequence patterns and arecalled the C1-set within the Ig superfamily. Other IgC domains fallwithin other sets. IgV domains also share sequence patterns and arecalled V set domains. IgV domains are longer than IgC domains andcontain an additional pair of β strands.

The term “PD-L1” refers to a specific PD-1 ligand. Two forms of humanPD-L1 molecules have been identified. One form is a naturally occurringPD-L1 soluble polypeptide, i.e., having a short hydrophilic domain atthe COOH-terminal end and no transmembrane domain, and is referred toherein as PD-L1S (shown in Table 1 as SEQ ID NO: 4). The second form isa cell-associated polypeptide, i.e., having a transmembrane andcytoplasmic domain, referred to herein as PD-L1M (shown in SEQ ID NO:6). The nucleic acid and amino acid sequences of representative humanPD-L1 biomarkers regarding PD-L1M are also available to the public atthe GenBank database under NM_014143.3 and NP_054862.1. PD-L1 proteinscomprise a signal sequence, and an IgV domain and an IgC domain. Thesignal sequence of SEQ ID NO: 4 is shown from about amino acid 1 toabout amino acid 18. The signal sequence of SEQ ID NO: 6 is shown: fromabout amino acid 1 to about amino acid 18. The IgV domain of SEQ ID NO:4 is shown from about amino acid 19 to about amino acid 134 and the IgVdomain of SEQ ID NO: 6 is shown from about amino acid 19 to about aminoacid 134. The IgC domain of SEQ ID NO: 4 is shown from about amino acid135 to about amino acid 227 and the IgC domain of SEQ ID NO: 6 is shownfrom about amino acid 135 to about amino acid 227. The hydrophilic tailof the PD-L1 exemplified in SEQ ID NO: 4 comprises a hydrophilic tailshown from about amino acid 228 to about amino acid 245. The PD-L1polypeptide exemplified in SEQ ID NO: 6 comprises a transmembrane domainshown from about amino acids 239 to about amino acid 259 of SEQ ID NO: 6and a cytoplasmic domain shown of about 30 amino acids from 260 to aboutamino acid 290 of SEQ ID NO: 6. In addition, nucleic acid andpolypeptide sequences of PD-L1 orthologs in organisms other than humansare well known and include, for example, mouse PD-L1 (NM_021893.3 andNP_068693.1), rat PD-L1 (NM_001191954.1 and NP_001178883.1), dog PD-L1(XM_541302.3 and XP_541302.3), cow PD-L1 (NM_001163412.1 andNP_001156884.1), and chicken PD-L1 (XM_424811.3 and XP_424811.3).

The term “TIM-3” refers to a type I cell-surface glycoprotein thatcomprises an N-terminal immunoglobulin (Ig)-like domain, a mucin domainwith O-linked glycosylations and with N-linked glycosylations close tothe membrane, a single transmembrane domain, and a cytoplasmic regionwith tyrosine phosphorylation motif(s) (see, for example, U.S. Pat.Publ. 2013/0156774). TIM-3 is a member of the T cell/transmembrane,immunoglobulin, and mucin (TIM) gene family. Nucleic acid andpolypeptide sequences of human TIM-3 are well known in the art and arepublicly available, for example, as described in NM_032782.4 andNP_116171.3. The term, as described above for useful markers such asPD-L1 and PD-1, encompasses any naturally occurring allelic, splicevariants, and processed forms thereof. Typically, TIM-3 refers to humanTIM-3 and can include truncated forms or fragments of the TIM-3polypeptide. In addition, nucleic acid and polypeptide sequences ofTIM-3 orthologs in organisms other than humans are well known andinclude, for example, mouse TIM-3 (NM_134250.2 and NP_599011.2),chimpanzee TIM-3 (XM_518059.4 and XP_518059.3), dog TIM-3(NM_001254715.1 and NP_001241644.1), cow TIM-3 (NM_001077105.2 andNP_001070573.1), and rat TIM-3 (NM_001100762.1 and NP_001094232.1). Inaddition, neutralizing anti-TIM-3 antibodies are well known in the art(see, at least U.S. Pat. Publ. 2013/0183688, Ngiow et al. (2011) CancerRes. 71:3540-3551; and antibody 344823 from R&D Biosystems, as well asclones 2C23, 5D12, 2E2, 4A4, and IG5, which are all published and thuspublicly available).

TIM-3 was originally identified as a mouse Th1-specific cell surfaceprotein that was expressed after several rounds of in vitro Th1differentiation, and was later shown to also be expressed on Th17 cells.In humans, TIM-3 is expressed on a subset of activated CD4+ T cells, ondifferentiated Th1 cells, on some CD8+ T cells, and at lower levels onTh17 cells (Hastings et al. (2009) Eur. J. Immunol. 39:2492-2501). TIM-3is also expressed on cells of the innate immune system including mousemast cells, subpopulations of macrophages and dendritic cells (DCs), NKand NKT cells, human monocytes, human dendritic cells, and on murineprimary bronchial epithelial cell lines. TIM-3 expression is regulatedby the transcription factor T-bet. TIM-3 can generate an inhibitorysignal resulting in apoptosis of Th1 and Tc1 cells, and can mediatephagocytosis of apoptotic cells and cross-presentation of antigen.Polymorphisms in TIM-1 and TIM-3 can reciprocally regulate the directionof T-cell responses (Freeman et al. (2010) Immunol. Rev. 235:172-89).

TIM-3 has several known ligands, including galectin-9,phosphatidylserine, and HMGB1. For example, galectin-9 is an S-typelectin with two distinct carbohydrate recognition domains joined by along flexible linker, and has an enhanced affinity for largerpoly-N-acetyllactosamine-containing structures. Galectin-9 does not havea signal sequence and is localized in the cytoplasm. However, it can besecreted and exerts its function by binding to glycoproteins on thetarget cell surface via their carbohydrate chains (Freeman et al. (2010)Immunol. Rev. 235:172-89). Engagement of TIM-3 by galectin-9 leads toTh1 cell death and a consequent decline in IFN-gamma. production. Whengiven in vivo, galectin-9 had beneficial effects in several murinedisease models, including an EAE model, a mouse model of arthritis, incardiac and skin allograft transplant models, and contacthypersensitivity and psoriatic models (Freeman et al. (2010) Immunol.Rev. 235:172-89). Residues important for TIM-3 binding to galectin-9include TIM-3(44), TIM-3(74), and TIM-3(100), which undergo N- and/or0-glycosylation. It is also known that TIM-3 mediates T-cell dysfunctionassociated with chronic viral infections (Golden-Mason et al. (2009) J.Virol. 83:9122-9130; Jones et al. (2008) J. Exp. Med. 205:2763-2779) andincreases HIV-1-specific T cell responses when blocked ex vivo(Golden-Mason et al. (2009) J. Virol. 83:9122-9130). In addition, inchronic HCV infection, TIM-3 expression was increased on CD4⁺ and CD8⁺ Tcells, specifically HCV-specific CD8+ cytotoxic T cells (CTLs) inchronic HCV infection and treatment with a blocking monoclonal antibodyto TIM-3 reversed HCV-specific T cell exhaustion (Jones et al. (2008) J.Exp. Med. 205:2763-2779).

The term “LAG-3,” also known as CD223, refers to a member of theimmunoglobulin supergene family and is structurally and geneticallyrelated to CD4 (see, U.S. Pat. Publ. 2011/0150892). LAG-3 is generallyknown as a membrane protein encoded by a gene located on the distal partof the short arm of chromosome 12, near the CD4 gene, suggesting thatthe LAG-3 gene may have evolved through gene duplication (Triebel et al.(1990) J. Exp. Med. 171:1393-1405). However, secreted forms of theprotein are known (e.g., for human and mouse TIM-3). Nucleic acid andpolypeptide sequences of human LAG-3 are well known in the art and arepublicly available, for example, as described in NM_002286.5 andNP_002277.4.

The term encompasses any naturally occurring allelic, splice variants,and processed forms thereof. Typically, LAG-3 refers to human LAG-3 andcan include truncated forms or fragments of the LAG-3 polypeptide. Inaddition, nucleic acid and polypeptide sequences of LAG-3 orthologs inorganisms other than humans are well known and include, for example,mouse LAG-3 (NM_008479.2 and NP_032505.1), chimpanzee LAG-3 (XM_508966.4and XP_508966.2), monkey LAG-3 (XM_001108923.2 and XP_001108923.1), cowLAG-3 (NM_00124949.1 and NP_001232878.1), rat LAG-3 (NM_212513.2 andNP_997678.2), and chicken LAG-3 (XM_416510.3, XP_416510.2,XM_004938117.1, and XP_004938174.1). In addition, neutralizinganti-LAG-3 antibodies are well known in the art (see, at least U.S. Pat.Publs. 2011/0150892 and 2010/0233183; Macon-Lemaitre and Triebel (2005)Immunology 115:170-178; Drake et al. (2006) J. Clin. Oncol. 24:2573;Richter et al. (2010) Int. Immunol. 22:13-23).

LAG-3 is not expressed on resting peripheral blood lymphocytes but isexpressed on activated T cells and NK cells and has a number offunctions (see, U.S. Pat. Publ. 2011/0150892). Similar to CD4, LAG-3 hasbeen demonstrated to interact with MHC Class II molecules but, unlikeCD4, LAG-3 does not interact with the human immunodeficiency virus gp120protein (Baixeras et al. (1992) J. Exp. Med. 176:327-337). Studies usinga soluble LAG-3 immunoglobulin fusion protein (sLAG-3Ig) demonstrateddirect and specific binding of LAG-3 to MHC class II on the cell surface(Huard et al. (1996) Eur. J. Immunol. 26:1180-1186). In in vitro studiesof antigen-specific T cell responses, the addition of anti-LAG-3antibodies led to increased T cell proliferation and higher expressionof activation antigens such as CD25, supporting a role for the LAG-/MHCclass II interaction in down-regulating antigen-dependent stimulation ofCD4+T lymphocytes (Huard et al. (1994) Eur. J. Immunol. 24:3216-3221).The intra-cytoplasmic region of LAG-3 has been demonstrated to interactwith a protein termed LAP, which is thought to be a signal transductionmolecule involved in the downregulation of the CD3/TCR activationpathway (Iouzalen et al. (2001) Eur. J. Immunol. 31:2885-2891).Furthermore, CD4+CD25+ regulatory T cells (T_(reg)) have been shown toexpress LAG-3 upon activation and antibodies to LAG-3 inhibitsuppression by induced regulatory T cells, both in vitro and in vivo,suggesting that LAG-3 contributes to the suppressor activity ofregulatory T cells (Huang et al. (2004) Immunity 21:503-513). Stillfurther, LAG-3 has been shown to negatively regulate T cell homeostasisby regulatory T cells in both T cell-dependent and independentmechanisms (Workman and Vignali (2005) J. Immunol. 174:688-695).

In certain circumstances, LAG-3 also has been shown to haveimmunostimulatory effects. For example, LAG-3 transfected tumor cellstransplanted into syngeneic mice showed marked growth reduction orcomplete regression as compared to untransfected tumor cells, suggestingthat LAG-3 expression on the tumor cells stimulated an anti-tumorresponse by triggering antigen presenting cells via MHC class IImolecules (Prigent et al. (1999) Eur. J. Immunol. 29:3867-3876).Additionally, soluble LAG-3 Ig fusion protein has been shown tostimulate both humoral and cellular immune responses when administeredto mice together with an antigen, indicating that soluble LAG-3Ig canfunction as a vaccine adjuvant (El Mir and Triebel (2000) J. Immunol.164:5583-5589). Furthermore, soluble human LAG-3Ig has been shown toamplify the in vitro generation of type I tumor-specific immunity(Casati et al. (2006) Cancer Res. 66:4450-4460). The functional activityof LAG-3 is reviewed further in Triebel (2003) Trends Immunol.24:619-622.

CTLA-4 is a T cell surface molecule that was originally identified bydifferential screening of a murine cytolytic T cell cDNA library, Brunetet al. (1987) Nature 328:267-270. The role of CTLA-4 as a secondreceptor for B7 is discussed in Linsley et al. (1991) J. Exp. Med.174:561-569. Freeman et al. (1993) Science 262:907-909 discusses CTLA-4in B7 deficient mice. Ligands for CTLA-4 are described in Lenschow etal. (1993) P.N.A.S. 90:11054-11058. Linsley et al. (1992) Science257:792-795 describes immunosuppression in vivo by a soluble form ofCTLA-4. Lenschow et al. (1992) Science 257:789-792 discusses long termsurvival of pancreatic islet grafts induced by CTLA-41g. It is suggestedin Walunas et al. (1994) Immunity 1:405-413, that CTLA-4 can function asa negative regulator of T cell activation. The amino acid and nucleotidesequence of CTLA-4 (e.g., human CTLA-4) are known in the art (e.g., asdescribed in U.S. Pat. Nos. 5,811,097 and 5,434,131, incorporated hereinby reference).

TABLE 1 SEQ ID NO: 1 Human PD-1 cDNA Sequencecactctggtg gggctgctcc aggc atg cag atc cca cag gcg ccc tgg cca  51                           Met Gln Ile Pro Gln Ala Pro Trp Pro                             1               5 gtc gtc tgg gcg gtg cta caa ctg ggc tgg cgg cca gga tgg ttc tta  99Val Val Trp Ala Val Leu Gln Leu Gly Trp Arg Pro Gly Trp Phe Leu  10                  15                  20                  25 gac tcc cca gac agg ccc tgg aac ccc ccc acc ttc tcc cca gcc ctg  147 Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu                  30                  35                  40 ctc gtg gtg acc gaa ggg gac aac gcc acc ttc acc tgc agc ttc tcc  195 Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser              45                  50                  55 aac aca tcg gag agc ttc gtg cta aac tgg tac cgc atg agc ccc agc  243 Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser          60                  65                  70 aac cag acg gac aag ctg gcc gcc ttc ccc gag gac cgc agc cag ccc  291 Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro      75                  80                  85 ggc cag gac tgc cgc ttc cgt gtc aca caa ctg ccc aac ggg cgt gac  339Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg Asp 90                   95                 100                 105 ttc cac atg agc gtg gtc agg gcc cgg cgc aat gac agc ggc acc tac  387 Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr                 110                 115                 120 ctc tgt ggg gcc atc tcc ctg gcc ccc aag gcg cag atc aaa gag agc  435Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu Ser             125                 130                 135 ctg cgg gca gag ctc agg gtg aca gag aga agg gca gaa gtg ccc aca  483Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro Thr         140                 145                 150 gcc cac ccc agc ccc tca ccc agg tca gcc ggc cag ttc caa acc ctg  531Ala His Pro Ser Pro Ser Pro Arg Ser Ala Gly Gln Phe Gln Thr Leu     155                 160                 165 gtg gtt ggt gtc gtg ggc ggc ctg ctg ggc agc ctg gtg ctg cta gtc  579Val Val Gly Val Val Gly Gly Leu Leu Gly Ser Leu Val Leu Leu Val 170                 175                 180                 185 tgg gtc ctg gcc gtc atc tgc tcc cgg gcc gca cga ggg aca ata gga  627Trp Val Leu Ala Val Ile Cys Ser Arg Ala Ala Arg Gly Thr Ile Gly                 190                 195                 200 gcc agg cgc acc ggc cag ccc ctg aag gag gac ccc tca gcc gtg cct  675Ala Arg Arg Thr Gly Gln Pro Leu Lys Glu Asp Pro Ser Ala Val Pro             205                 210                 215 gtg ttc tct gtg gac tat ggg gag ctg gat ttc cag tgg cga gag aag  723Val Phe Ser Val Asp Tyr Gly Glu Leu Asp Phe Gln Trp Arg Glu Lys         220                 225                 230 acc ccg gag ccc ccc gtg ccc tgt gtc cct gag cag acg gag tat gcc 771Thr Pro Glu Pro Pro Val Pro Cys Val Pro Glu Gln Thr Glu Tyr Ala     235                 240                 245 acc att gtc ttt cct agc gga atg ggc acc tca tcc ccc gcc cgc agg 819Thr Ile Val Phe Pro Ser Gly Met Gly Thr Ser Ser Pro Ala Arg Arg 250                 255                 260                 265 ggc tca gct gac ggc cct cgg agt gcc cag cca ctg agg cct gag gat 867Gly Ser Ala Asp Gly Pro Arg Ser Ala Gln Pro Leu Arg Pro Glu Asp                 270                 275                 280gga cac tgc tct tgg ccc ctc tgaccggctt ccttggccac cagtgttctg cag 921Gly His Cys Ser Trp Pro Leu              285 SEQ ID NO: 2 Human PD-1 Amino Acid Sequence Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln   1               5                  10                  15 Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp             20                  25                  30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp          35                  40                  45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val      50                  55                  60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala  65                  70                  75                  80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg                  85                  90                  95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg             100                 105                 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu         115                 120                 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val     130                 135                 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145                 150                 155                 160Arg Ser Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly                 165                 170                 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys             180                 185                 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro         195                 200                 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly     210                 215                 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro 225                 230                 235                 240 Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly                 245                 250                 255 Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg             260                 265                 270 Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu         275                 280                 285 SEQ ID NO: 3 Human PD-L1S cDNA Acid Sequence gcttcccgag gctccgcacc agccgcgctt ctgtccgcct gcagggcatt ccagaaag   58atg agg ata ttt gct gtc ttt ata ttc atg acc tac tgg cat ttg ctg 106 Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu   1               5                  10                  15 aac gca ttt act gtc acg gtt ccc aag gac cta tat gtg gta gag tat 154 Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr              20                  25                  30 ggt agc aat atg aca att gaa tgc aaa ttc cca gta gaa aaa caa tta  202Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu          35                  40                  45 gac ctg gct gca cta att gtc tat tgg gaa atg gag gat aag aac att  250 Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile      50                  55                  60 att caa ttt gtg cat gga gag gaa gac ctg aag gtt cag cat agt agc  298 Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser  65                  70                  75                  80 tac aga cag agg gcc cgg ctg ttg aag gac cag ctc tcc ctg gga aat  346 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn                  85                  90                  95 gct gca ctt cag atc aca gat gtg aaa ttg cag gat gca ggg gtg tac  394 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr             100                 105                 110 cgc tgc atg atc agc tat ggt ggt gcc gac tac aag cga att act gtg  442 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val         115                 120                 125 aaa gtc aat gcc cca tac aac aaa atc aac caa aga att ttg gtt gtg  490 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val     130                 135                 140 gat cca gtc acc tct gaa cat gaa ctg aca tgt cag gct gag ggc tac  538 Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145                 150                 155                 160 ccc aag gcc gaa gtc atc tgg aca agc agt gac cat caa gtc ctg agt  586 Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser                 165                 170                 175 ggt aag acc acc acc acc aat tcc aag aga gag gag aag ctt ttc aat  634 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn             180                 185                 190 gtg acc agc aca ctg aga atc aac aca aca act aat gag att ttc tac  682 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr         195                 200                 205 tgc act ttt agg aga tta gat cct gag gaa aac cat aca gct gaa ttg  730 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu     210                 215                 220 gtc atc cca ggt aat att ctg aat gtg tcc att aaa ata tgt cta aca  778 Val Ile Pro Gly Asn Ile Leu Asn Val Ser Ile Lys Ile Cys Leu Thr 225                 230                 235                 240 ctg tcc cct agc acc tagcatgatg tctgcctatc atagtcattc agtgattgtt  833 Leu Ser Pro Ser Thr                  245 gaataaatga atgaatgaat aacactatgt ttacaaaata tatcctaatt cctcacctcc  893 attcatccaa accatattgt tacttaataa acattcagca gatatttatg gaataaaaaa  953 aaaaaaaaaa aaaaa  968  SEQ ID NO: 4 Human PD-L1S Amino Acid Sequence Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu   1               5                  10                  15 Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr             20                  25                  30 Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu          35                  40                  45 Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile      50                  55                  60 Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser  65                  70                  75                  80 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn                  85                  90                  95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr             100                 105                 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val         115                 120                 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val     130                 135                 140 Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145                 150                 155                 160 Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser                 165                 170                 175 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn             180                 185                 190 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr         195                 200                 205 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu     210                 215                 220 Val Ile Pro Gly Asn Ile Leu Asn Val Ser Ile Lys Ile Cys Leu Thr 225                 230                 235                 240 Leu Ser Pro Ser Thr                  245 SEQ ID NO: 5 Human PD-L1M cDNA Acid Sequence cgaggctccg caccagccgc gcttctgtcc gcctgcaggg cattccagaa agatgagg   58                                                        Met Arg                                                               1 ata ttt gct gtc ttt ata ttc atg acc tac tgg cat ttg ctg aac gca   106 Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu Asn Ala           5                  10                  15 ttt act gtc acg gtt ccc aag gac cta tat gtg gta gag tat ggt agc   154 Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser      20                  25                  30 aat atg aca att gaa tgc aaa ttc cca gta gaa aaa caa tta gac ctg  202Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu35                   40                  45                 50 gct gca cta att gtc tat tgg gaa atg gag gat aag aac att att caa  250Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln                  55                  60                  65 ttt gtg cat gga gag gaa gac ctg aag gtt cag cat agt agc tac aga    298Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg              70                  75                  80 cag agg gcc cgg ctg ttg aag gac cag ctc tcc ctg gga aat gct gca    346Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala          85                  90                  95 ctt cag atc aca gat gtg aaa ttg cag gat gca ggg gtg tac cgc tgc    394Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys     100                 105                 110 atg atc agc tat ggt ggt gcc gac tac aag cga att act gtg aaa gtc    442Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val 115                 120                 125                 130 aat gcc cca tac aac aaa atc aac caa aga att ttg gtt gtg gat cca    490Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro                 135                 140                 145 gtc acc tct gaa cat gaa ctg aca tgt cag gct gag ggc tac ccc aag    538Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys             150                 155                 160 gcc gaa gtc atc tgg aca agc agt gac cat caa gtc ctg agt ggt aag    586Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys         165                 170                 175 acc acc acc acc aat tcc aag aga gag gag aag ctt ttc aat gtg acc    634Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr     180                 185                 190 agc aca ctg aga atc aac aca aca act aat gag att ttc tac tgc act    682Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr 195                 200                 205                 210 ttt agg aga tta gat cct gag gaa aac cat aca gct gaa ttg gtc atc    730Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile                 215                 220                 225 cca gaa cta cct ctg gca cat cct cca aat gaa agg act cac ttg gta    778Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His Leu Val             230                 235                 240 att ctg gga gcc atc tta tta tgc ctt ggt gta gca ctg aca ttc atc    826Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr Phe Ile         245                 250                 255 ttc cgt tta aga aaa ggg aga atg atg gat gtg aaa aaa tgt ggc atc    874Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys Gly Ile     260                 265                 270 caa gat aca aac tca aag aag caa agt gat aca cat ttg gag gag acg    922Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu Glu Thr 275                 280                 285                 290 taatccagca ttggaacttc tgatcttcaa gcagggattc tcaacctgtg gtttaggggt    982tcatcggggc tgagcgtgac aagaggaagg aatgggcccg tgggatgcag gcaatgtggg   1042acttaaaagg cccaagcact gaaaatggaa cctggcgaaa gcagaggagg agaatgaaga   1102aagatggagt caaacaggga gcctggaggg agaccttgat actttcaaat gcctgagggg   1162ctcatcgacg cctgtgacag ggagaaagga tacttctgaa caaggagcct ccaagcaaat   1222catccattgc tcatcctagg aagacgggtt gagaatccct aatttgaggg tcagttcctg   1282cagaagtgcc ctttgcctcc actcaatgcc tcaatttgtt ttctgcatga ctgagagtct   1342cagtgttgga acgggacagt atttatgtat gagtttttcc tatttatttt gagtctgtga   1402ggtcttcttg tcatgtgagt gtggttgtga atgatttctt ttgaagatat attgtagtag   1462atgttacaat tttgtcgcca aactaaactt gctgcttaat gatttgctca catctagtaa   1522aacatggagt atttgtaaaa aaaaaaaaaa a   1553SEQ ID NO: 6 Human PD-L1M Amino Acid SequenceMet Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu   1               5                  10                  15 Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr              20                  25                  30 Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu          35                  40                  45 Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile      50                  55                  60 Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser  65                  70                  75                  80 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn                  85                  90                  95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr             100                 105                 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val         115                 120                 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val     130                 135                 140 Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145                 150                 155                 160 Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser                 165                 170                 175 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn             180                 185                 190 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr         195                 200                 205 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu     210                 215                 220 Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His 225                 230                 235                 240 Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr                 245                 250                 255 Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys             260                 265                 270 Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu         275                 280                 285  Glu Thr      290 SEQ ID NO: 7 Mouse PD-L1 cDNA Sequence    1  atgaggatat ttgctggcat tatattcaca gcctgctgtc acttgctacg ggcgtttact  61  atcacggctc caaaggactt gtacgtggtg gagtatggca gcaacgtcac gatggagtgc 121agattccctg tagaacggga gctggacctg cttgcgttag tggtgtactg ggaaaaggaa  181gatgagcaag tgattcagtt tgtggcagga gaggaggacc ttaagcctca gcacagcaac  241ttcaggggga gagcctcgct gccaaaggac cagcttttga agggaaatgc tgcccttcag  301atcacagacg tcaagctgca ggacgcaggc gtttactgct gcataatcag ctacggtggt  361gcggactaca agcgaatcac gctgaaagtc aatgccccat accgcaaaat caaccagaga  421atttccgtgg atccagccac ttctgagcat gaactaatat gtcaggccga gggttatcca  481gaagctgagg taatctggac aaacagtgac caccaacccg tgagtgggaa gagaagtgtc  541accacttccc ggacagaggg gatgcttctc aatgtgacca gcagtctgag ggtcaacgcc  601acagcgaatg atgttttcta ctgtacgttt tggagatcac agccagggca aaaccacaca  661gcggagctga tcatcccaga actgcctgca acacatcctc cacagaacag gactcactgg  721gtgcttctgg gatccatcct gttgttcctc attgtagtgt ccacggtcct cctcttcttg  781agaaaacaag tgagaatgct agatgtggag aaatgtggcg ttgaagatac aagctcaaaa  841aaccgaaatg atacacaatt cgaggagacg taa SEQ ID NO: 8 Mouse PD-L1 Amino Acid Sequence    1mrifagiift acchllraft itapkdlyvv eygsnvtmec rfpvereldl lalvvyweke   61deqviqfvag eedlkpqhsn frgraslpkd qllkgnaalq itdvklqdag vycciisygg  121adykritlkv napyrkinqr isvdpatseh elicqaegyp eaeviwtnsd hqpvsgkrsv  181ttsrtegmll nvtsslrvna tandvfyctf wrsqpgqnht aeliipelpa thppqnrthw  241vllgsillfl ivvstvllfl rkqvrmldve kcgvedtssk nrndtqfeet SEQ ID NO: 9 Human TIM-3 cDNA Sequence   1atgttttcac atcttccctt tgactgtgtc ctgctgctgc tgctgctact acttacaagg   61tcctcagaag tggaatacag agcggaggtc ggtcagaatg cctatctgcc ctgcttctac  121accccagccg ccccagggaa cctcgtgccc gtctgctggg gcaaaggagc ctgtcctgtg  181tttgaatgtg gcaacgtggt gctcaggact gatgaaaggg atgtgaatta ttggacatcc  241agatactggc taaatgggga tttccgcaaa ggagatgtgt ccctgaccat agagaatgtg  301actctagcag acagtgggat ctactgctgc cggatccaaa tcccaggcat aatgaatgat  361gaaaaattta acctgaagtt ggtcatcaaa ccagccaagg tcacccctgc accgactcgg  421cagagagact tcactgcagc ctttccaagg atgcttacca ccaggggaca tggcccagca  481gagacacaga cactggggag cctccctgat ataaatctaa cacaaatatc cacattggcc  541aatgagttac gggactctag attggccaat gacttacggg actctggagc aaccatcaga  601ataggcatct acatcggagc agggatctgt gctgggctgg ctctggctct tatcttcggc  661gctttaattt tcaaatggta ttctcatagc aaagagaaga tacagaattt aagcctcatc  721tctttggcca acctccctcc ctcaggattg gcaaatgcag tagcagaggg aattcgctca  781gaagaaaaca tctataccat tgaagagaac gtatatgaag tggaggagcc caatgagtat  841tattgctatg tcagcagcag gcagcaaccc tcacaacctt tgggttgtcg ctttgcaatg  901ccatag  SEQ ID NO: 10 Human TIM-3 Amino Acid Sequence    1mfshlpfdcv lllllllltr sseveyraev gqnaylpcfy tpaapgnlvp vcwgkgacpv  61fecgnvvlrt derdvnywts rywlngdfrk gdvsltienv tladsgiycc riqipgimnd  121ekfnlklvik pakvtpaptr qrdftaafpr mlttrghgpa etqtlgslpd initqistla  181nelrdsrlan dlrdsgatir igiyigagic aglalalifg alifkwyshs kekiqnlsli  241slanlppsgl anavaegirs eeniytieen vyeveepney ycyvssrqqp sqplgcrfam  301 pSEQ ID NO: 11 Mouse TIM-3 cDNA Sequence    1atgttttcag gtcttaccct caactgtgtc ctgctgctgc tgcaactact acttgcaagg   61tcattggaaa atgcttatgt gtttgaggtt ggtaagaatg cctatctgcc ctgcagttac  121actctatcta cacctggggc acttgtgcct atgtgctggg gcaagggatt ctgtccttgg  181tcacagtgta ccaacgagtt gctcagaact gatgaaagaa atgtgacata tcagaaatcc  241agcagatacc agctaaaggg cgatctcaac aaaggagacg tgtctctgat cataaagaat  301gtgactctgg atgaccatgg gacctactgc tgcaggatac agttccctgg tcttatgaat  361gataaaaaat tagaactgaa attagacatc aaagcagcca aggtcactcc agctcagact  421gcccatgggg actctactac agcttctcca agaaccctaa ccacggagag aaatggttca  481gagacacaga cactggtgac cctccataat aacaatggaa caaaaatttc cacatgggct  541gatgaaatta aggactctgg agaaacgatc agaactgcta tccacattgg agtgggagtc  601tctgctgggt tgaccctggc acttatcatt ggtgtcttaa tccttaaatg gtattcctgt  661aagaaaaaga agttatcgag tttgagcctt attacactgg ccaacttgcc tccaggaggg  721ttggcaaatg caggagcagt caggattcgc tctgaggaaa atatctacac catcgaggag  781aacgtatatg aagtggagaa ttcaaatgag tactactgct acgtcaacag ccagcagcca  841tcctga SEQ ID NO: 12 Mouse TIM-3 Amino Acid Sequence    1mfsgltlncv llllqlllar slenayvfev gknaylpcsy tlstpgalvp mcwgkgfcpw   61sqctnellrt dernvtyqks sryqlkgdln kgdvsliikn vtlddhgtyc criqfpglmn  121dkklelkldi kaakvtpaqt ahgdsttasp rtltterngs etqtivtlhn nngtkistwa  181deikdsgeti rtaihigvgv sagltlalii gvlilkwysc kkkklsslsl itlanlppgg  241lanagavrir seeniytiee nvyevensne yycyvnsqqp s SEQ ID NO: 13 Human LAG-3 cDNA Sequence     1atgtgggagg ctcagttcct gggcttgctg tttctgcagc cgctttgggt ggctccagtg    61aagcctctcc agccaggggc tgaggtcccg gtggtgtggg cccaggaggg ggctcctgcc   121cagctcccct gcagccccac aatccccctc caggatctca gccttctgcg aagagcaggg   181gtcacttggc agcatcagcc agacagtggc ccgcccgctg ccgcccccgg ccatcccctg   241gcccccggcc ctcacccggc ggcgccctcc tcctgggggc ccaggccccg ccgctacacg   301gtgctgagcg tgggtcccgg aggcctgcgc agcgggaggc tgcccctgca gccccgcgtc   361cagctggatg agcgcggccg gcagcgcggg gacttctcgc tatggctgcg cccagcccgg   421cgcgcggacg ccggcgagta ccgcgccgcg gtgcacctca gggaccgcgc cctctcctgc   481cgcctccgtc tgcgcctggg ccaggcctcg atgactgcca gccccccagg atctctcaga   541gcctccgact gggtcatttt gaactgctcc ttcagccgcc ctgaccgccc agcctctgtg   601cattggttcc ggaaccgggg ccagggccga gtccctgtcc gggagtcccc ccatcaccac   661ttagcggaaa gcttcctctt cctgccccaa gtcagcccca tggactctgg gccctggggc   721tgcatcctca cctacagaga tggcttcaac gtctccatca tgtataacct cactgttctg   781ggtctggagc ccccaactcc cttgacagtg tacgctggag caggttccag ggtggggctg   841ccctgccgcc tgcctgctgg tgtggggacc cggtctttcc tcactgccaa gtggactcct   901cctgggggag gccctgacct cctggtgact ggagacaatg gcgactttac ccttcgacta   961gaggatgtga gccaggccca ggctgggacc tacacctgcc atatccatct gcaggaacag  1021cagctcaatg ccactgtcac attggcaatc atcacagtga ctcccaaatc ctttgggtca  1081cctggatccc tggggaagct gctttgtgag gtgactccag tatctggaca agaacgcttt  1141gtgtggagct ctctggacac cccatcccag aggagtttct caggaccttg gctggaggca  1201caggaggccc agctcctttc ccagccttgg caatgccagc tgtaccaggg ggagaggctt  1261cttggagcag cagtgtactt cacagagctg tctagcccag gtgcccaacg ctctgggaga  1321gccccaggtg ccctcccagc aggccacctc ctgctgtttc tcatccttgg tgtcctttct  1381ctgctccttt tggtgactgg agcctttggc tttcaccttt ggagaagaca gtggcgacca  1441agacgatttt ctgccttaga gcaagggatt caccctccgc aggctcagag caagatagag  1501gagctggagc aagaaccgga gccggagccg gagccggaac cggagcccga gcccgagccc  1561gagccggagc agctctga  SEQ ID NO: 14 Human LAG-3 Amino Acid Sequence    1mweaqflgll flqplwvapv kplqpgaevp vvwagegapa qlpcsptipl qdlsllrrag   61vtwqhqpdsg ppaaapghpl apgphpaaps swgprprryt vlsvgpgglr sgrlplqpry  121qldergrqrg dfslwlrpar radageyraa vhlrdralsc rlrlrlgqas mtasppgslr  181asdwvilncs fsrpdrpasv hwfrnrgqgr vpvresphhh laesflflpq vspmdsgpwg  241ciltyrdgfn vsimynitvl glepptpltv yagagsrvgl perlpagvgt rsfltakwtp  301pgggpdllvt gdngdftlrl edvsqaqagt ytchihlqeq qlnatvtlai itvtpksfgs  361pgslgkllce vtpvsgqerf vwssldtpsq rsfsgpwlea qeaqllsqpw qcqlyggerl  421lgaavyftel sspgaqrsgr apgalpaghl llflilgvls llllvtgafg fhlwrrqwrp  481rrfsaleqgi hppqaqskie eleqepepep epepepepep epeql SEQ ID NO: 15 Mouse LAG-3 cDNA Sequence     1atgagggagg acctgctcct tggctttttg cttctgggac tgctttggga agctccagtt    61gtgtcttcag ggcctgggaa agagctcccc gtggtgtggg cccaggaggg agctcccgtc   121catcttccct gcagcctcaa atcccccaac ctggatccta actttctacg aagaggaggg   181gttatctggc aacatcaacc agacagtggc caacccactc ccatcccggc ccttgacctt   241caccagggga tgccctcgcc tagacaaccc gcacccggtc gctacacggt gctgagcgtg   301gctccaggag gcctgcgcag cgggaggcag cccctgcatc cccacgtgca gctggaggag   361cgcggcctcc agcgcgggga cttctctctg tggttgcgcc cagctctgcg caccgatgcg   421ggcgagtacc acgccaccgt gcgcctcccg aaccgcgccc tctcctgcag tctccgcctg   481cgcgtcggcc aggcctcgat gattgctagt ccctcaggag tcctcaagct gtctgattgg   541gtccttttga actgctcctt cagccgtcct gaccgcccag tctctgtgca ctggttccag   601ggccagaacc gagtgcctgt ctacaactca ccgcgtcatt ttttagctga aactttcctg   661ttactgcccc aagtcagccc cctggactct gggacctggg gctgtgtcct cacctacaga   721gatggcttca atgtctccat cacgtacaac ctcaaggttc tgggtctgga gcccgtagcc   781cctctgacag tgtacgctgc tgaaggttct agggtggagc tgccctgtca tttgccccca   841ggagtgggga ccccttcttt gctcattgcc aagtggactc ctcctggagg aggtcctgag   901ctccccgtgg ctggaaagag tggcaatttt acccttcacc ttgaggctgt gggtctggca   961caggctggga cctacacctg tagcatccat ctgcagggac agcagctcaa tgccactgtc  1021acgttggcgg tcatcacagt gactcccaaa tccttcgggt tacctggctc ccgggggaag  1081ctgttgtgtg aggtaacccc ggcatctgga aaggaaagat ttgtgtggcg tcccctgaac  1141aatctgtcca ggagttgccc gggccctgtg ctggagattc aggaggccag gctccttgct  1201gagcgatggc agtgtcagct gtacgagggc cagaggcttc ttggagcgac agtgtacgcc  1261gcagagtcta gctcaggcgc ccacagtgct aggagaatct caggtgacct taaaggaggc  1321catctcgttc tcgttctcat ccttggtgcc ctctccctgt tccttttggt ggccggggcc  1381tttggctttc actggtggag aaaacagttg ctactgagaa gattttctgc cttagaacat  1441gggattcagc catttccggc tcagaggaag atagaggagc tggagcgaga actggagacg  1501gagatgggac aggagccgga gcccgagccg gagccacagc tggagccaga gcccaggcag  1561ctctga  SEQ ID NO: 16 Mouse LAG-3 Amino Acid Sequence    1mredlllgfl llgllweapv vssgpgkelp vvwagegapv hlpcslkspn ldpnflrrgg   61viwqhqpdsg qptpipaldl hqgmpsprqp apgrytvlsv apgglrsgrq plhphvqlee  121rglqrgdfsl wlrpalrtda geyhatvrlp nralscslrl rvgqasmias psgvlklsdw  181vllncsfsrp drpvsvhwfq gqnrvpvyns prhflaetfl llpqvsplds gtwgcvltyr  241dgfnvsityn lkvlglepva pltvyaaegs rvelpchlpp gvgtpsllia kwtppgggpe  301lpvagksgnf tlhleavgla qagtytcsih lqgqqlnatv tlavitvtpk sfglpgsrgk  361llcevtpasg kerfvwrpln nlsrscpgpv leiqearlla erwqcqlyeg qrllgatvya  421aesssgahsa rrisgdlkgg hlvlvlilga lslfllvaga fgfhwwrkql llrrfsaleh  481giqpfpaqrk ieelerelet emgqepepep epqlepeprq l 

Inhibitors of immune checkpoint modulators are known in the art, and canbe used in any of the methods described herein. For example, anti-PD-1antibodies or soluble polypeptide inhibitors can be used. In someembodiments, the anti-PD-1 antibody is chosen from MDX-1106, Merck 3475or CT-011. MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558 orNivolumab. In some embodiments, the anti-PD-1 antibody is Nivolumab (CASRegistry Number: 946414-94-4). Nivolumab (also referred to as BMS-936558or MDX1106; Bristol-Myers Squibb) is a fully human IgG4 monoclonalantibody which specifically blocks PD1. Nivolumab (clone 5C4) and otherhuman monoclonal antibodies that specifically bind to PD1 are disclosedin U.S. Pat. No. 8,008,449 and WO2006/121168. Lambrolizumab (alsoreferred to as MK03475; Merck) is a humanized IgG4 monoclonal antibodythat binds to PD1. Lambrolizumab and other humanized anti-PD1 antibodiesare disclosed in U.S. Pat. No. 8,354,509 and WO2009/114335. Pidilizumab(CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that bindsto PD1. Pidilizumab and other humanized anti-PD1 monoclonal antibodiesare disclosed in WO2009/101611. Other anti-PD1 antibodies include AMP514 (Amplimmune), among others, e.g., anti-PD1 antibodies disclosed inU.S. Pat. No. 8,609,089, US 2010028330, and/or US 20120114649. In someembodiments, the PD-1 inhibitor is an immunoadhesin (e.g., animmunoadhesin comprising an extracellular or PD-1 binding portion ofPD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of animmunoglobulin sequence). In some embodiments, the PD-1 inhibitor isAMP-224. AMP-224 (B7-DCIg; Amplimmune; e.g., disclosed in WO2010/027827and WO2011/066342), is a PD-L2 Fc fusion soluble receptor that blocksthe interaction between PD1 and B7-H1.

In other embodiments, anti-PD-L1 antibodies or soluble polypeptideinhibitors can be used. In some embodiments, the PD-L1 inhibitor isanti-PD-L1 antibody. In other embodiments, the anti-PD-L1 bindingantagonist is chosen from YW243.55.570, MPDL3280A or MDX-1105. MDX-1105,also known as BMS-936559, is an anti-PD-L1 antibody described inWO2007/005874. MDPL3280A (Genentech/Roche) is a human Fc optimized IgG1monoclonal antibody that binds to PD-L1. MDPL3280A and other humanmonoclonal antibodies to PD-L1 are disclosed in U.S. Pat. No. 7,943,743and U.S Publication No.: 20120039906. Antibody YW243.55.570 (heavy andlight chain variable region sequences shown in SEQ ID Nos. 20 and 21,respectively) is an anti-PD-L1 described in WO 2010/077634.

Exemplary anti-CTLA4 antibodies that can be used in the methodsdisclosed herein include, but are not limited to, Tremelimumab (IgG2monoclonal antibody available from Pfizer, formerly known asticilimumab, CP-675,206); and Ipilimumab (CTLA-4 antibody, also known asMDX-010, CAS No. 477202-00-9). Antibodies to T cell costimulatorymolecules such as CTLA-4 (e.g., U.S. Pat. No. 5,811,097), CTLA-4inhibitor (e.g., CP-675,206, ipilimumab)

II. Methods of Treating Hematologic Cancers

a. Agents Useful for Treating Hematologic Cancers

It is demonstrated herein that inhibiting or blocking a function of PD-1or PD-L1 and TIM-3, LAG-3 or CTLA-4 synergistically and significantlyblocks the establishment and progression of malignancies (e.g.,hematologic cancers such as multiple myeloma) in all subjects analyzed.Inhibition or blockade of PD-1 or PD-L1 and TIM-3, LAG-3 or CTLA-4function can block the establishment and progression of similarmalignancies. Thus, the agents of the present invention described hereinthat modulate the interaction between, for example, PD-1 or PD-L1 andTIM-3, LAG-3 or CTLA-4, whether directly or indirectly, can upregulateor downregulate the immune system and, thereby, upregulate ordownregulate an immune response.

PD-1, PD-L1 and TIM-3, LAG-3 and CTLA-4 are immune checkpoint regulatorsthat deliver co-inhibitory immune signals. Thus, in one embodiment,agents that neutralize an activity of PD-1 or PD-L1 and TIM-3, LAG-3 orCTLA-4 can prevent inhibitory signaling and upregulate an immuneresponse. In another embodiment, agents which directly block theinteraction between PD-1 or PD-L1 and its natural receptor(s), andTIM-3, LAG-3 or CTLA-4 and its natural receptor(s) (e.g., blockingantibodies) can prevent inhibitory signaling and upregulate an immuneresponse. Alternatively, agents that indirectly block the interactionbetween PD-1 or PD-L1 and its natural receptor(s), and TIM-3, LAG-3 orCTLA-4 and its natural receptor(s) can prevent inhibitory signaling andupregulate an immune response. For example, soluble B7-1 or solublePD-1, by binding to a PD-L1 polypeptide indirectly reduces the effectiveconcentration of PD-L1 polypeptide available to bind to theimmunoinhibitor receptor, PD-1. Exemplary agents for upregulating animmune response include antibodies against PD-1, PD-L1, LAG-3, CTLA-4and/or TIM-3 that block the interaction between the immune checkpointregulator and its natural receptor(s); a non-activating form of PD-1,PD-L1, LAG-3, CTLA-4 and/or TIM-3 (e.g., a dominant negativepolypeptide), small molecules or peptides that block the interactionbetween the immune checkpoint regulator and its natural receptor(s);fusion proteins (e.g. the extracellular portion of −1, PD-L1, LAG-3,CTLA-4 or TIM-3 fused to the Fc portion of an antibody orimmunoglobulin) that bind to their natural receptor(s); nucleic acidmolecules that block PD-1, PD-L1, LAG-3, CTLA-4 and/or TIM-3 geneexpression, e.g., transcription or translation; and the like.

Additional agents useful in the methods of the present invention includeantibodies, small molecules, peptides, peptidomimetics, natural ligands,and derivatives of natural ligands, that can either bind and/orinactivate or inhibit protein biomarkers of the invention, including thebiomarkers listed in Table 1, or fragments thereof; as well as RNAinterference, antisense, nucleic acid aptamers, etc. that candownregulate the expression and/or activity of the biomarkers of theinvention, including the biomarkers listed in Table 1, or fragmentsthereof.

In one embodiment, isolated nucleic acid molecules that specificallyhybridize with or encode one or more biomarkers of the invention, listedin Table 1 for example, or biologically active portions thereof. As usedherein, the term “nucleic acid molecule” is intended to include DNAmolecules (i.e., cDNA or genomic DNA) and RNA molecules (i.e., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA. An “isolated” nucleic acid moleculeis one which is separated from other nucleic acid molecules which arepresent in the natural source of the nucleic acid. Preferably, an“isolated” nucleic acid is free of sequences which naturally flank thenucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolatednucleic acid molecules corresponding to the one or more biomarkerslisted in Table 1 or described herein can contain less than about 5 kb,4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived (i.e., a lymphoma cell).Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule,can be substantially free of other cellular material, or culture mediumwhen produced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of one or more biomarkers listedin Table 1 or a nucleotide sequence which is at least about 50%,preferably at least about 60%, more preferably at least about 70%, yetmore preferably at least about 80%, still more preferably at least about90%, and most preferably at least about 95% or more (e.g., about 98%)homologous to the nucleotide sequence of one or more biomarkers listedin Table 1 or a portion thereof (i.e., 100, 200, 300, 400, 450, 500, ormore nucleotides), can be isolated using standard molecular biologytechniques and the sequence information provided herein. For example, ahuman cDNA can be isolated from a human cell line using all or portionof the nucleic acid molecule, or fragment thereof, as a hybridizationprobe and standard hybridization techniques (i.e., as described inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Moreover, anucleic acid molecule encompassing all or a portion of the nucleotidesequence of one or more biomarkers listed in Table 1 or a nucleotidesequence which is at least about 50%, preferably at least about 60%,more preferably at least about 70%, yet more preferably at least about80%, still more preferably at least about 90%, and most preferably atleast about 95% or more homologous to the nucleotide sequence, orfragment thereof, can be isolated by the polymerase chain reaction usingoligonucleotide primers designed based upon the sequence of the one ormore biomarkers listed in Table 1, or fragment thereof, or thehomologous nucleotide sequence. For example, mRNA can be isolated frommuscle cells (i.e., by the guanidinium-thiocyanate extraction procedureof Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and cDNA can beprepared using reverse transcriptase (i.e., Moloney MLV reversetranscriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reversetranscriptase, available from Seikagaku America, Inc., St. Petersburg,Fla.). Synthetic oligonucleotide primers for PCR amplification can bedesigned according to well-known methods in the art. A nucleic acid ofthe invention can be amplified using cDNA or, alternatively, genomicDNA, as a template and appropriate oligonucleotide primers according tostandard PCR amplification techniques. The nucleic acid so amplified canbe cloned into an appropriate vector and characterized by DNA sequenceanalysis. Furthermore, oligonucleotides corresponding to the nucleotidesequence of one or more biomarkers listed in Table 1 can be prepared bystandard synthetic techniques, i.e., using an automated DNA synthesizer.

Probes based on the nucleotide sequences of one or more biomarkerslisted in Table 1 can be used to detect or confirm the desiredtranscripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, i.e., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which express one or more biomarkers listed in Table 1, such asby measuring a level of one or more biomarkers nucleic acid in a sampleof cells from a subject, i.e., detecting mRNA levels of one or morebiomarkers listed in Table 1.

Nucleic acid molecules encoding proteins corresponding to one or morebiomarkers listed in Table 1, or portions thereof, from differentspecies are also contemplated. For example, rat or monkey cDNA can beidentified based on the nucleotide sequence of a human and/or mousesequence and such sequences are well known in the art. In oneembodiment, the nucleic acid molecule(s) of the invention encodes aprotein or portion thereof which includes an amino acid sequence whichis sufficiently homologous to an amino acid sequence of one or morebiomarkers listed in Table 1, such that the protein or portion thereofmodulates (e.g., enhance), one or more of the following biologicalactivities: a) binding to the biomarker; b) modulating the copy numberof the biomarker; c) modulating the expression level of the biomarker;and d) modulating the activity level of the biomarker.

As used herein, the language “sufficiently homologous” refers toproteins or portions thereof which have amino acid sequences whichinclude a minimum number of identical or equivalent (e.g., an amino acidresidue which has a similar side chain as an amino acid residue in oneor more biomarkers listed in Table 1, or fragment thereof) amino acidresidues to an amino acid sequence of the biomarker, or fragmentthereof, such that the protein or portion thereof modulates (e.g.,enhance) one or more of the following biological activities: a) bindingto the biomarker; b) modulating the copy number of the biomarker; c)modulating the expression level of the biomarker; and d) modulating theactivity level of the biomarker.

In another embodiment, the protein is at least about 50%, preferably atleast about 60%, more preferably at least about 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to theentire amino acid sequence of the biomarker, or a fragment thereof.

Portions of proteins encoded by nucleic acid molecules of the one ormore biomarkers listed in Table 1 are preferably biologically activeportions of the protein. As used herein, the term “biologically activeportion” of one or more biomarkers listed in Table 1 is intended toinclude a portion, e.g., a domain/motif, that has one or more of thebiological activities of the full-length protein.

Standard binding assays, e.g., immunoprecipitations and yeast two-hybridassays, as described herein, or functional assays, e.g., RNAi oroverexpression experiments, can be performed to determine the ability ofthe protein or a biologically active fragment thereof to maintain abiological activity of the full-length protein.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence of the one or more biomarkers listed inTable 1, or fragment thereof due to degeneracy of the genetic code andthus encode the same protein as that encoded by the nucleotide sequence,or fragment thereof. In another embodiment, an isolated nucleic acidmolecule of the invention has a nucleotide sequence encoding a proteinhaving an amino acid sequence of one or more biomarkers listed in Table1, or fragment thereof, or a protein having an amino acid sequence whichis at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more homologous to the amino acid sequence of the oneor more biomarkers listed in Table 1, or fragment thereof. In anotherembodiment, a nucleic acid encoding a polypeptide consists of nucleicacid sequence encoding a portion of a full-length fragment of interestthat is less than 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145,140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, or 70amino acids in length.

It will be appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequences of theone or more biomarkers listed in Table 1 may exist within a population(e.g., a mammalian and/or human population). Such genetic polymorphismsmay exist among individuals within a population due to natural allelicvariation. As used herein, the terms “gene” and “recombinant gene” referto nucleic acid molecules comprising an open reading frame encoding oneor more biomarkers listed in Table 1, preferably a mammalian, e.g.,human, protein. Such natural allelic variations can typically result in1-5% variance in the nucleotide sequence of the one or more biomarkerslisted in Table 1. Any and all such nucleotide variations and resultingamino acid polymorphisms in the one or more biomarkers listed in Table 1that are the result of natural allelic variation and that do not alterthe functional activity of the one or more biomarkers listed in Table 1are intended to be within the scope of the invention. Moreover, nucleicacid molecules encoding one or more biomarkers listed in Table 1 fromother species.

In addition to naturally-occurring allelic variants of the one or morebiomarkers listed in Table 1 that may exist in the population, theskilled artisan will further appreciate that changes can be introducedby mutation into the nucleotide sequence, or fragment thereof, therebyleading to changes in the amino acid sequence of the encoded one or morebiomarkers listed in Table 1, without altering the functional ability ofthe one or more biomarkers listed in Table 1. For example, nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues can be made in the sequence, or fragment thereof. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of the one or more biomarkers listed in Table 1without altering the activity of the one or more biomarkers listed inTable 1, whereas an “essential” amino acid residue is required for theactivity of the one or more biomarkers listed in Table 1. Other aminoacid residues, however, (e.g., those that are not conserved or onlysemi-conserved between mouse and human) may not be essential foractivity and thus are likely to be amenable to alteration withoutaltering the activity of the one or more biomarkers listed in Table 1.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. Preferably, the alignment can be performed using the ClustalMethod. Multiple alignment parameters include GAP Penalty=10, Gap LengthPenalty=10. For DNA alignments, the pairwise alignment parameters can beHtuple=2, Gap penalty=5, Window=4, and Diagonal saved=4. For proteinalignments, the pairwise alignment parameters can be Ktuple=1, Gappenalty=3, Window=5, and Diagonals Saved=5.

In a preferred embodiment, the percent identity between two amino acidsequences is determined using the Needleman and Wunsch (J. Mol. Biol.(48):444-453 (1970)) algorithm which has been incorporated into the GAPprogram in the GCG software package (available online), using either aBlossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12,10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yetanother preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (available online), using a NWSgapdna.CMP matrix and agap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4,5, or 6. In another embodiment, the percent identity between two aminoacid or nucleotide sequences is determined using the algorithm of E.Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has beenincorporated into the ALIGN program (version 2.0) (available online),using a PAM120 weight residue table, a gap length penalty of 12 and agap penalty of 4.

An isolated nucleic acid molecule encoding a protein homologous to oneor more biomarkers listed in Table 1, or fragment thereof, can becreated by introducing one or more nucleotide substitutions, additionsor deletions into the nucleotide sequence, or fragment thereof, or ahomologous nucleotide sequence such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or more predictednon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). Thus, a predicted nonessential amino acidresidue in one or more biomarkers listed in Table 1 is preferablyreplaced with another amino acid residue from the same side chainfamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of the coding sequence of the oneor more biomarkers listed in Table 1, such as by saturation mutagenesis,and the resultant mutants can be screened for an activity describedherein to identify mutants that retain desired activity. Followingmutagenesis, the encoded protein can be expressed recombinantlyaccording to well-known methods in the art and the activity of theprotein can be determined using, for example, assays described herein.

The levels of one or more biomarkers listed in Table 1 may be assessedby any of a wide variety of well-known methods for detecting expressionof a transcribed molecule or protein. Non-limiting examples of suchmethods include immunological methods for detection of proteins, proteinpurification methods, protein function or activity assays, nucleic acidhybridization methods, nucleic acid reverse transcription methods, andnucleic acid amplification methods.

In preferred embodiments, the levels of one or more biomarkers listed inTable 1 are ascertained by measuring gene transcript (e.g., mRNA), by ameasure of the quantity of translated protein, or by a measure of geneproduct activity. Expression levels can be monitored in a variety ofways, including by detecting mRNA levels, protein levels, or proteinactivity, any of which can be measured using standard techniques.Detection can involve quantification of the level of gene expression(e.g., genomic DNA, cDNA, mRNA, protein, or enzyme activity), or,alternatively, can be a qualitative assessment of the level of geneexpression, in particular in comparison with a control level. The typeof level being detected will be clear from the context.

In a particular embodiment, the mRNA expression level can be determinedboth by in situ and by in vitro formats in a biological sample usingmethods known in the art. The term “biological sample” is intended toinclude tissues, cells, biological fluids and isolates thereof, isolatedfrom a subject, as well as tissues, cells and fluids present within asubject. Many expression detection methods use isolated RNA. For invitro methods, any RNA isolation technique that does not select againstthe isolation of mRNA can be utilized for the purification of RNA fromcells (see, e.g., Ausubel et al., ed., Current Protocols in MolecularBiology, John Wiley & Sons, New York 1987-1999). Additionally, largenumbers of tissue samples can readily be processed using techniques wellknown to those of skill in the art, such as, for example, thesingle-step RNA isolation process of Chomczynski (1989, U.S. Pat. No.4,843,155).

The isolated mRNA can be used in hybridization or amplification assaysthat include, but are not limited to, Southern or Northern analyses,polymerase chain reaction analyses and probe arrays. One preferreddiagnostic method for the detection of mRNA levels involves contactingthe isolated mRNA with a nucleic acid molecule (probe) that canhybridize to the mRNA encoded by the gene being detected. The nucleicacid probe can be, for example, a full-length cDNA, or a portionthereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250or 500 nucleotides in length and sufficient to specifically hybridizeunder stringent conditions to a mRNA or genomic DNA encoding one or morebiomarkers listed in Table 1. Other suitable probes for use in thediagnostic assays of the invention are described herein. Hybridizationof an mRNA with the probe indicates that one or more biomarkers listedin Table 1 is being expressed.

In one format, the mRNA is immobilized on a solid surface and contactedwith a probe, for example by running the isolated mRNA on an agarose geland transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probe(s) are immobilizedon a solid surface and the mRNA is contacted with the probe(s), forexample, in a gene chip array, e.g., an Affymetrix™ gene chip array. Askilled artisan can readily adapt known mRNA detection methods for usein detecting the level of the one or more biomarkers listed in Table 1.

An alternative method for determining mRNA expression level in a sampleinvolves the process of nucleic acid amplification, e.g., by RT-PCR (theexperimental embodiment set forth in Mullis, 1987, U.S. Pat. No.4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci.USA, 88:189-193), self-sustained sequence replication (Guatelli et al.,1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptionalamplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No.5,854,033) or any other nucleic acid amplification method, followed bythe detection of the amplified molecules using techniques well-known tothose of skill in the art. These detection schemes are especially usefulfor the detection of nucleic acid molecules if such molecules arepresent in very low numbers. As used herein, amplification primers aredefined as being a pair of nucleic acid molecules that can anneal to 5′or 3′ regions of a gene (plus and minus strands, respectively, orvice-versa) and contain a short region in between. In general,amplification primers are from about 10 to 30 nucleotides in length andflank a region from about 50 to 200 nucleotides in length. Underappropriate conditions and with appropriate reagents, such primerspermit the amplification of a nucleic acid molecule comprising thenucleotide sequence flanked by the primers.

For in situ methods, mRNA does not need to be isolated from the cellsprior to detection. In such methods, a cell or tissue sample isprepared/processed using known histological methods. The sample is thenimmobilized on a support, typically a glass slide, and then contactedwith a probe that can hybridize to the one or more biomarkers listed inTable 1.

As an alternative to making determinations based on the absoluteexpression level, determinations may be based on the normalizedexpression level of one or more biomarkers listed in Table 1. Expressionlevels are normalized by correcting the absolute expression level bycomparing its expression to the expression of a non-biomarker gene,e.g., a housekeeping gene that is constitutively expressed. Suitablegenes for normalization include housekeeping genes such as the actingene, or epithelial cell-specific genes. This normalization allows thecomparison of the expression level in one sample, e.g., a subjectsample, to another sample, e.g., a normal sample, or between samplesfrom different sources.

The level or activity of a protein corresponding to one or morebiomarkers listed in Table 1 can also be detected and/or quantified bydetecting or quantifying the expressed polypeptide. The polypeptide canbe detected and quantified by any of a number of means well known tothose of skill in the art. These may include analytic biochemicalmethods such as electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, and the like, or variousimmunological methods such as fluid or gel precipitin reactions,immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, Western blotting, and the like. A skilledartisan can readily adapt known protein/antibody detection methods foruse in determining whether cells express the biomarker of interest.

The present invention further provides soluble, purified and/or isolatedpolypeptide forms of one or more biomarkers listed in Table 1, orfragments thereof. In addition, it is to be understood that any and allattributes of the polypeptides described herein, such as percentageidentities, polypeptide lengths, polypeptide fragments, biologicalactivities, antibodies, etc. can be combined in any order or combinationwith respect to any biomarker listed in Table 1 and combinationsthereof.

In one aspect, a polypeptide may comprise a full-length amino acidsequence corresponding to one or more biomarkers listed in Table 1 or afull-length amino acid sequence with 1 to about 20 conservative aminoacid substitutions. An amino acid sequence of any described herein canalso be at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 99.5% identical to the full-length sequence of one ormore biomarkers listed in Table 1, which is either described herein,well known in the art, or a fragment thereof. In another aspect, thepresent invention contemplates a composition comprising an isolatedpolypeptide corresponding to one or more biomarkers listed in Table 1and less than about 25%, or alternatively 15%, or alternatively 5%,contaminating biological macromolecules or polypeptides.

The present invention further provides compositions related toproducing, detecting, or characterizing such polypeptides, or fragmentthereof, such as nucleic acids, vectors, host cells, and the like. Suchcompositions may serve as compounds that modulate the expression and/oractivity of one or more biomarkers described herein or, for example,listed in Table 1.

An isolated polypeptide or a fragment thereof (or a nucleic acidencoding such a polypeptide) corresponding to one or more biomarkers ofthe invention, including the biomarkers listed in Table 1 or fragmentsthereof, can be used as an immunogen to generate antibodies that bind tosaid immunogen, using standard techniques for polyclonal and monoclonalantibody preparation according to well-known methods in the art. Anantigenic peptide comprises at least 8 amino acid residues andencompasses an epitope present in the respective full length moleculesuch that an antibody raised against the peptide forms a specific immunecomplex with the respective full length molecule. Preferably, theantigenic peptide comprises at least 10 amino acid residues. In oneembodiment such epitopes can be specific for a given polypeptidemolecule from one species, such as mouse or human (i.e., an antigenicpeptide that spans a region of the polypeptide molecule that is notconserved across species is used as immunogen; such non conservedresidues can be determined using an alignment such as that providedherein).

In one embodiment, an antibody binds substantially specifically to PD-L1and inhibits or blocks its immunoinhibitory function, such as byinterrupting its interaction with an inhibitory receptor like PD-1. Inanother embodiment, an antibody binds substantially specifically toTIM-3 and inhibits or blocks its immunoinhibitory function, such as byinterrupting its interaction with galectin-9 or phosphatidylserine.

For example, a polypeptide immunogen typically is used to prepareantibodies by immunizing a suitable subject (e.g., rabbit, goat, mouseor other mammal) with the immunogen. An appropriate immunogenicpreparation can contain, for example, a recombinantly expressed orchemically synthesized molecule or fragment thereof to which the immuneresponse is to be generated. The preparation can further include anadjuvant, such as Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic preparation induces a polyclonal antibody response to theantigenic peptide contained therein.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a polypeptide immunogen. The polypeptide antibodytiter in the immunized subject can be monitored over time by standardtechniques, such as with an enzyme linked immunosorbent assay (ELISA)using immobilized polypeptide. If desired, the antibody directed againstthe antigen can be isolated from the mammal (e.g., from the blood) andfurther purified by well-known techniques, such as protein Achromatography, to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique (originally described by Kohler and Milstein (1975)Nature 256:495-497) (see also Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al.(1976) Proc. Natl. Acad. Sci. 76:2927-31; Yeh et al. (1982) Int. J.Cancer 29:269-75), the more recent human B cell hybridoma technique(Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique(Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generallyKenneth, R. H. in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); Lerner, E. A.(1981) Yale J. Biol. Med. 54:387-402; Gefter, M. L. et al. (1977)Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typicallya myeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with an immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds to the polypeptideantigen, preferably specifically.

Any of the many well-known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating amonoclonal antibody against one or more biomarkers of the invention,including the biomarkers listed in Table 1, or a fragment thereof (see,e.g., Galfre, G. et al. (1977) Nature 266:55052; Gefter et al. (1977)supra; Lerner (1981) supra; Kenneth (1980) supra). Moreover, theordinary skilled worker will appreciate that there are many variationsof such methods which also would be useful. Typically, the immortal cellline (e.g., a myeloma cell line) is derived from the same mammalianspecies as the lymphocytes. For example, murine hybridomas can be madeby fusing lymphocytes from a mouse immunized with an immunogenicpreparation of the present invention with an immortalized mouse cellline. Preferred immortal cell lines are mouse myeloma cell lines thatare sensitive to culture medium containing hypoxanthine, aminopterin andthymidine (“HAT medium”). Any of a number of myeloma cell lines can beused as a fusion partner according to standard techniques, e.g., theP3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O—Ag14 myeloma lines. Thesemyeloma lines are available from the American Type Culture Collection(ATCC), Rockville, Md. Typically, HAT-sensitive mouse myeloma cells arefused to mouse splenocytes using polyethylene glycol (“PEG”). Hybridomacells resulting from the fusion are then selected using HAT medium,which kills unfused and unproductively fused myeloma cells (unfusedsplenocytes die after several days because they are not transformed).Hybridoma cells producing a monoclonal antibody of the invention aredetected by screening the hybridoma culture supernatants for antibodiesthat bind a given polypeptide, e.g., using a standard ELISA assay.

As an alternative to preparing monoclonal antibody-secreting hybridomas,a monoclonal specific for one of the above described polypeptides can beidentified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) withthe appropriate polypeptide to thereby isolate immunoglobulin librarymembers that bind the polypeptide. Kits for generating and screeningphage display libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening an antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al.International Publication No. WO 92/18619; Dower et al. InternationalPublication No. WO 91/17271; Winter et al. International Publication WO92/20791; Markland et al. International Publication No. WO 92/15679;Breitling et al. International Publication WO 93/01288; McCafferty etal. International Publication No. WO 92/01047; Garrard et al.International Publication No. WO 92/09690; Ladner et al. InternationalPublication No. WO 90/02809; Fuchs et al. (1991) Biotechnology (NY)9:1369-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J.12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clarkson etal. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci.USA 89:3576-3580; Garrard et al. (1991) Biotechnology (NY) 9:1373-1377;Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137; Barbas et al.(1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et al.(1990) Nature 348:552-554.

Since it is well known in the art that antibody heavy and light chainCDR3 domains play a particularly important role in the bindingspecificity/affinity of an antibody for an antigen, the recombinantmonoclonal antibodies of the present invention prepared as set forthabove preferably comprise the heavy and light chain CDR3s of variableregions of the antibodies described herein and well known in the art.Similarly, the antibodies can further comprise the CDR2s of variableregions of said antibodies. The antibodies can further comprise theCDR1s of variable regions of said antibodies. In other embodiments, theantibodies can comprise any combinations of the CDRs.

The CDR1, 2, and/or 3 regions of the engineered antibodies describedabove can comprise the exact amino acid sequence(s) as those of variableregions of the present invention disclosed herein. However, theordinarily skilled artisan will appreciate that some deviation from theexact CDR sequences may be possible while still retaining the ability ofthe antibody to bind a desired target, such as PD-L1, TIM-3, or LAG-3effectively (e.g., conservative sequence modifications). Accordingly, inanother embodiment, the engineered antibody may be composed of one ormore CDRs that are, for example, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to one or moreCDRs of the present invention described herein or otherwise publiclyavailable.

The structural features of non-human or human antibodies (e.g., a ratanti-mouse/anti-human PD-L1 antibody) can be used to create structurallyrelated human antibodies that retain at least one functional property ofthe antibodies of the present invention, such as binding to PD-L1,TIM-3, or LAG-3. Another functional property includes inhibiting bindingof the original known, non-human or human antibodies in a competitionELISA assay.

In some embodiments, monoclonal antibodies capable of binding andinhibiting/blocking PD-L1, TIM-3, and/or LAG-3 are provided, comprisinga heavy chain wherein the variable domain comprises at least a CDRhaving a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical from the group of heavychain variable domain CDRs presented herein or otherwise publiclyavailable.

Similarly, monoclonal antibodies binding and inhibiting/blocking PD-L1,TIM-3, and/or LAG-3, comprising a light chain wherein the variabledomain comprises at least a CDR having a sequence that is at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%identical from the group of light chain variable domain CDRs presentedherein or otherwise publicly available, are also provided.

Monoclonal antibodies capable of binding and inhibiting/blocking PD-L1,TIM-3, and/or LAG-3, comprising a heavy chain wherein the variabledomain comprises at least a CDR having a sequence that is at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%identical from the group of heavy chain variable domain CDRs presentedherein or otherwise publicly available; and comprising a light chainwherein the variable domain comprises at least a CDR having a sequencethat is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.5% or 100% identical from the group of light chain variabledomain CDRs presented herein or otherwise publicly available, are alsoprovided.

A skilled artisan will note that such percentage homology is equivalentto and can be achieved by introducing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore conservative amino acid substitutions within a given CDR.

The monoclonal antibodies of the present invention can comprise a heavychain, wherein the variable domain comprises at least a CDR having asequence selected from the group consisting of the heavy chain variabledomain CDRs presented herein or otherwise publicly available and a lightchain, wherein the variable domain comprises at least a CDR having asequence selected from the group consisting of the light chain variabledomain CDRs presented herein or otherwise publicly available.

Such monoclonal antibodies can comprise a light chain, wherein thevariable domain comprises at least a CDR having a sequence selected fromthe group consisting of CDR-L1, CDR-L2, and CDR-L3, as described herein;and/or a heavy chain, wherein the variable domain comprises at least aCDR having a sequence selected from the group consisting of CDR-H1,CDR-H2, and CDR-H3, as described herein. In some embodiments, themonoclonal antibodies capable of binding human Gall comprises orconsists of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3, asdescribed herein.

The heavy chain variable domain of the monoclonal antibodies of thepresent invention can comprise or consist of the vH amino acid sequenceset forth herein or otherwise publicly available and/or the light chainvariable domain of the monoclonal antibodies of the present inventioncan comprise or consist of the vκ amino acid sequence set forth hereinor otherwise publicly available.

The present invention further provides fragments of said monoclonalantibodies which include, but are not limited to, Fv, Fab, F(ab′)2,Fab′, dsFv, scFv, sc(Fv)2 and diabodies; and multispecific antibodiesformed from antibody fragments.

Other fragments of the monoclonal antibodies of the present inventionare also contemplated. For example, individual immunoglobulin heavyand/or light chains are provided, wherein the variable domains thereofcomprise at least a CDR presented herein or otherwise publiclyavailable. In one embodiment, the immunoglobulin heavy chain comprisesat least a CDR having a sequence that is at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical from thegroup of heavy chain or light chain variable domain CDRs presentedherein or otherwise publicly available. In another embodiment, animmunoglobulin light chain comprises at least a CDR having a sequencethat is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.5% or 100% identical from the group of light chain or heavychain variable domain CDRs presented herein or otherwise publiclyavailable, are also provided.

In some embodiments, the immunoglobulin heavy and/or light chaincomprises a variable domain comprising at least one of CDR-L1, CDR-L2,CDR-L3, CDR-H1, CDR-H2, or CDR-H3 described herein. Such immunoglobulinheavy chains can comprise or consist of at least one of CDR-H1, CDR-H2,and CDR-H3. Such immunoglobulin light chains can comprise or consist ofat least one of CDR-L1, CDR-L2, and CDR-L3.

In other embodiments, an immunoglobulin heavy and/or light chainaccording to the present invention comprises or consists of a vH or vκvariable domain sequence, respectively, provided herein or otherwisepublicly available.

The present invention further provides polypeptides which have asequence selected from the group consisting of vH variable domain, vκvariable domain, CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3sequences described herein.

Antibodies, immunoglobulins, and polypeptides of the invention can beused in an isolated (e.g., purified) form or contained in a vector, suchas a membrane or lipid vesicle (e.g. a liposome).

Amino acid sequence modification(s) of the antibodies described hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody. Itis known that when a humanized antibody is produced by simply graftingonly CDRs in VH and VL of an antibody derived from a non-human animal inFRs of the VH and VL of a human antibody, the antigen binding activityis reduced in comparison with that of the original antibody derived froma non-human animal. It is considered that several amino acid residues ofthe VH and VL of the non-human antibody, not only in CDRs but also inFRs, are directly or indirectly associated with the antigen bindingactivity. Hence, substitution of these amino acid residues withdifferent amino acid residues derived from FRs of the VH and VL of thehuman antibody would reduce binding activity and can be corrected byreplacing the amino acids with amino acid residues of the originalantibody derived from a non-human animal.

Modifications and changes may be made in the structure of the antibodiesdescribed herein, and in the DNA sequences encoding them, and stillobtain a functional molecule that encodes an antibody and polypeptidewith desirable characteristics. For example, certain amino acids may besubstituted by other amino acids in a protein structure withoutappreciable loss of activity. Since the interactive capacity and natureof a protein define the protein's biological functional activity,certain amino acid substitutions can be made in a protein sequence, and,of course, in its DNA encoding sequence, while nevertheless obtaining aprotein with like properties. It is thus contemplated that variouschanges may be made in the antibodies sequences of the invention, orcorresponding DNA sequences which encode said polypeptides, withoutappreciable loss of their biological activity.

In making the changes in the amino sequences of polypeptide, thehydropathic index of amino acids may be considered. The importance ofthe hydropathic amino acid index in conferring interactive biologicfunction on a protein is generally understood in the art. It is acceptedthat the relative hydropathic character of the amino acid contributes tothe secondary structure of the resultant protein, which in turn definesthe interaction of the protein with other molecules, for example,enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophane (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (<RTI 3.5); asparagine (−3.5); lysine (−3.9); andarginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

Another type of amino acid modification of the antibody of the inventionmay be useful for altering the original glycosylation pattern of theantibody to, for example, increase stability. By “altering” is meantdeleting one or more carbohydrate moieties found in the antibody, and/oradding one or more glycosylation sites that are not present in theantibody. Glycosylation of antibodies is typically N-linked. “N-linked”refers to the attachment of the carbohydrate moiety to the side chain ofan asparagine residue. The tripeptide sequences asparagine-X-serine andasparagines-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of the carbohydratemoiety to the asparagine side chain. Thus, the presence of either ofthese tripeptide sequences in a polypeptide creates a potentialglycosylation site. Addition of glycosylation sites to the antibody isconveniently accomplished by altering the amino acid sequence such thatit contains one or more of the above-described tripeptide sequences (forN-linked glycosylation sites). Another type of covalent modificationinvolves chemically or enzymatically coupling glycosides to theantibody. These procedures are advantageous in that they do not requireproduction of the antibody in a host cell that has glycosylationcapabilities for N- or O-linked glycosylation. Depending on the couplingmode used, the sugar(s) may be attached to (a) arginine and histidine,(b) free carboxyl groups, (c) free sulfhydryl groups such as those ofcysteine, (d) free hydroxyl groups such as those of serine, threonine,orhydroxyproline, (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan, or (f) the amide group of glutamine. Forexample, such methods are described in WO87/05330.

Similarly, removal of any carbohydrate moieties present on the antibodymay be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described by Sojahr et al.(1987) and by Edge et al. (1981). Enzymatic cleavage of carbohydratemoieties on antibodies can be achieved by the use of a variety of endo-and exo-glycosidases as described by Thotakura et al. (1987).

Other modifications can involve the formation of immunoconjugates. Forexample, in one type of covalent modification, antibodies or proteinsare covalently linked to one of a variety of non proteinaceous polymers,e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

Conjugation of antibodies or other proteins of the present inventionwith heterologous agents can be made using a variety of bifunctionalprotein coupling agents including but not limited to N-succinimidyl(2-pyridyldithio) propionate (SPDP), succinimidyl(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT),bifunctional derivatives of imidoesters (such as dimethyl adipimidateHCL), active esters (such as disuccinimidyl suberate), aldehydes (suchas glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, carbon labeled1-isothiocyanatobenzyl methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody (WO 94/11026).

In another aspect, the present invention features antibodies conjugatedto a therapeutic moiety, such as a cytotoxin, a drug, and/or aradioisotope. When conjugated to a cytotoxin, these antibody conjugatesare referred to as “immunotoxins.” A cytotoxin or cytotoxic agentincludes any agent that is detrimental to (e.g., kills) cells. Examplesinclude taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). An antibody of the presentinvention can be conjugated to a radioisotope, e.g., radioactive iodine,to generate cytotoxic radiopharmaceuticals for treating a relateddisorder, such as a cancer.

Conjugated antibodies, in addition to therapeutic utility, can be usefulfor diagnostically or prognostically to monitor polypeptide levels intissue as part of a clinical testing procedure, e.g., to determine theefficacy of a given treatment regimen. Detection can be facilitated bycoupling (i e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate (FITC),rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride orphycoerythrin (PE); an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S, or ³H. [0134] As used herein, the term“labeled”, with regard to the antibody, is intended to encompass directlabeling of the antibody by coupling (i.e., physically linking) adetectable substance, such as a radioactive agent or a fluorophore (e.g.fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine(Cy5)) to the antibody, as well as indirect labeling of the antibody byreactivity with a detectable substance.

The antibody conjugates of the present invention can be used to modify agiven biological response. The therapeutic moiety is not to be construedas limited to classical chemical therapeutic agents. For example, thedrug moiety may be a protein or polypeptide possessing a desiredbiological activity. Such proteins may include, for example, anenzymatically active toxin, or active fragment thereof, such as abrin,ricin A, Pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor or interferon-.gamma.; or, biological responsemodifiers such as, for example, lymphokines, interleukin-1 (“IL-1”),interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophagecolony stimulating factor (“GM-CSF”), granulocyte colony stimulatingfactor (“G-CSF”), or other cytokines or growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243 56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623 53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303 16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119 58 (1982).

In some embodiments, conjugations can be made using a “cleavable linker”facilitating release of the cytotoxic agent or growth inhibitory agentin a cell. For example, an acid-labile linker, peptidase-sensitivelinker, photolabile linker, dimethyl linker or disulfide-containinglinker (See e.g. U.S. Pat. No. 5,208,020) may be used. Alternatively, afusion protein comprising the antibody and cytotoxic agent or growthinhibitory agent may be made, by recombinant techniques or peptidesynthesis. The length of DNA may comprise respective regions encodingthe two portions of the conjugate either adjacent one another orseparated by a region encoding a linker peptide which does not destroythe desired properties of the conjugate.

Additionally, recombinant polypeptide antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Patent Publication PCT/US86/02269; Akira et al. EuropeanPatent Application 184,187; Taniguchi, M. European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT Application WO 86/01533; Cabilly et al. U.S. Pat. No.4,816,567; Cabilly et al. European Patent Application 125,023; Better etal. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad.Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sunet al. (1987) Proc. Natl. Acad. Sci. 84:214-218; Nishimura et al. (1987)Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw etal. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985)Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter U.S.Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan etal. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-4060.

In addition, humanized antibodies can be made according to standardprotocols such as those disclosed in U.S. Pat. No. 5,565,332. In anotherembodiment, antibody chains or specific binding pair members can beproduced by recombination between vectors comprising nucleic acidmolecules encoding a fusion of a polypeptide chain of a specific bindingpair member and a component of a replicable generic display package andvectors containing nucleic acid molecules encoding a second polypeptidechain of a single binding pair member using techniques known in the art,e.g., as described in U.S. Pat. Nos. 5,565,332, 5,871,907, or 5,733,743.The use of intracellular antibodies to inhibit protein function in acell is also known in the art (see e.g., Carlson, J. R. (1988) Mol.Cell. Biol. 8:2638-2646; Biocca, S. et al. (1990) EMBO J. 9:101-108;Werge, T. M. et al. (1990) FEBS Lett. 274:193-198; Carlson, J. R. (1993)Proc. Natl. Acad. Sci. USA 90:7427-7428; Marasco, W. A. et al. (1993)Proc. Natl. Acad. Sci. USA 90:7889-7893; Biocca, S. et al. (1994)Biotechnology (NY) 12:396-399; Chen, S-Y. et al. (1994) Hum. Gene Ther.5:595-601; Duan, L et al. (1994) Proc. Natl. Acad. Sci. USA91:5075-5079; Chen, S-Y. et al. (1994) Proc. Natl. Acad. Sci. USA91:5932-5936; Beerli, R. R. et al. (1994) J. Biol. Chem.269:23931-23936; Beerli, R. R. et al. (1994) Biochem. Biophys. Res.Commun. 204:666-672; Mhashilkar, A. M. et al. (1995) EMBO J.14:1542-1551; Richardson, J. H. et al. (1995) Proc. Natl. Acad. Sci. USA92:3137-3141; PCT Publication No. WO 94/02610 by Marasco et al.; and PCTPublication No. WO 95/03832 by Duan et al.).

Additionally, fully human antibodies could be made against biomarkers ofthe invention, including the biomarkers listed in Table 1, or fragmentsthereof. Fully human antibodies can be made in mice that are transgenicfor human immunoglobulin genes, e.g. according to Hogan, et al.,“Manipulating the Mouse Embryo: A Laboratory Manuel,” Cold Spring HarborLaboratory. Briefly, transgenic mice are immunized with purifiedimmunogen. Spleen cells are harvested and fused to myeloma cells toproduce hybridomas. Hybridomas are selected based on their ability toproduce antibodies which bind to the immunogen. Fully human antibodieswould reduce the immunogenicity of such antibodies in a human.

In one embodiment, an antibody for use in the instant invention is abispecific or multispecific antibody. A bispecific antibody has bindingsites for two different antigens within a single antibody polypeptide.Antigen binding may be simultaneous or sequential. Triomas and hybridhybridomas are two examples of cell lines that can secrete bispecificantibodies. Examples of bispecific antibodies produced by a hybridhybridoma or a trioma are disclosed in U.S. Pat. No. 4,474,893.Bispecific antibodies have been constructed by chemical means (Staerz etal. (1985) Nature 314:628, and Perez et al. (1985) Nature 316:354) andhybridoma technology (Staerz and Bevan (1986) Proc. Natl. Acad. Sci.USA, 83:1453, and Staerz and Bevan (1986) Immunol. Today 7:241).Bispecific antibodies are also described in U.S. Pat. No. 5,959,084.Fragments of bispecific antibodies are described in U.S. Pat. No.5,798,229.

Bispecific agents can also be generated by making heterohybridomas byfusing hybridomas or other cells making different antibodies, followedby identification of clones producing and co-assembling both antibodies.They can also be generated by chemical or genetic conjugation ofcomplete immunoglobulin chains or portions thereof such as Fab and Fvsequences. The antibody component can bind to a polypeptide or afragment thereof of one or more biomarkers of the invention, includingone or more biomarkers listed in Table 1, or a fragment thereof. In oneembodiment, the bispecific antibody could specifically bind to both apolypeptide or a fragment thereof and its natural binding partner(s) ora fragment(s) thereof.

In another aspect of this invention, peptides or peptide mimetics can beused to antagonize the activity of one or more biomarkers of theinvention, including one or more biomarkers listed in Table 1, or afragment(s) thereof. In one embodiment, variants of one or morebiomarkers listed in Table 1 which function as a modulating agent forthe respective full length protein, can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, forantagonist activity. In one embodiment, a variegated library of variantsis generated by combinatorial mutagenesis at the nucleic acid level andis encoded by a variegated gene library. A variegated library ofvariants can be produced, for instance, by enzymatically ligating amixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential polypeptide sequences is expressible asindividual polypeptides containing the set of polypeptide sequencestherein. There are a variety of methods which can be used to producelibraries of polypeptide variants from a degenerate oligonucleotidesequence. Chemical synthesis of a degenerate gene sequence can beperformed in an automatic DNA synthesizer, and the synthetic gene thenligated into an appropriate expression vector. Use of a degenerate setof genes allows for the provision, in one mixture, of all of thesequences encoding the desired set of potential polypeptide sequences.Methods for synthesizing degenerate oligonucleotides are known in theart (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al.(1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.

In addition, libraries of fragments of a polypeptide coding sequence canbe used to generate a variegated population of polypeptide fragments forscreening and subsequent selection of variants of a given polypeptide.In one embodiment, a library of coding sequence fragments can begenerated by treating a double stranded PCR fragment of a polypeptidecoding sequence with a nuclease under conditions wherein nicking occursonly about once per polypeptide, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA which can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with S1 nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal, C-terminal and internal fragments of various sizes of thepolypeptide.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of polypeptides. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify variants ofinterest (Arkin and Youvan (1992) Proc. Natl. Acad. Sci. USA89:7811-7815; Delagrave et al. (1993) Protein Eng. 6(3):327-331). In oneembodiment, cell based assays can be exploited to analyze a variegatedpolypeptide library. For example, a library of expression vectors can betransfected into a cell line which ordinarily synthesizes one or morebiomarkers of the invention, including one or more biomarkers listed inTable 1, or a fragment thereof. The transfected cells are then culturedsuch that the full length polypeptide and a particular mutantpolypeptide are produced and the effect of expression of the mutant onthe full length polypeptide activity in cell supernatants can bedetected, e.g., by any of a number of functional assays. Plasmid DNA canthen be recovered from the cells which score for inhibition, oralternatively, potentiation of full length polypeptide activity, and theindividual clones further characterized.

Systematic substitution of one or more amino acids of a polypeptideamino acid sequence with a D-amino acid of the same type (e.g., D-lysinein place of L-lysine) can be used to generate more stable peptides. Inaddition, constrained peptides comprising a polypeptide amino acidsequence of interest or a substantially identical sequence variation canbe generated by methods known in the art (Rizo and Gierasch (1992) Annu.Rev. Biochem. 61:387, incorporated herein by reference); for example, byadding internal cysteine residues capable of forming intramoleculardisulfide bridges which cyclize the peptide.

The amino acid sequences disclosed herein will enable those of skill inthe art to produce polypeptides corresponding peptide sequences andsequence variants thereof. Such polypeptides can be produced inprokaryotic or eukaryotic host cells by expression of polynucleotidesencoding the peptide sequence, frequently as part of a largerpolypeptide. Alternatively, such peptides can be synthesized by chemicalmethods. Methods for expression of heterologous proteins in recombinanthosts, chemical synthesis of polypeptides, and in vitro translation arewell known in the art and are described further in Maniatis et al.Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold SpringHarbor, N.Y.; Berger and Kimmel, Methods in Enzymology, Volume 152,Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., SanDiego, Calif.; Merrifield, J. (1969) J. Am. Chem. Soc. 91:501; ChaikenI. M. (1981) CRC Crit. Rev. Biochem. 11: 255; Kaiser et al. (1989)Science 243:187; Merrifield, B. (1986) Science 232:342; Kent, S. B. H.(1988) Annu. Rev. Biochem. 57:957; and Offord, R. E. (1980)Semisynthetic Proteins, Wiley Publishing, which are incorporated hereinby reference).

Peptides can be produced, typically by direct chemical synthesis.Peptides can be produced as modified peptides, with nonpeptide moietiesattached by covalent linkage to the N-terminus and/or C-terminus. Incertain preferred embodiments, either the carboxy-terminus or theamino-terminus, or both, are chemically modified. The most commonmodifications of the terminal amino and carboxyl groups are acetylationand amidation, respectively. Amino-terminal modifications such asacylation (e.g., acetylation) or alkylation (e.g., methylation) andcarboxy-terminal-modifications such as amidation, as well as otherterminal modifications, including cyclization, can be incorporated intovarious embodiments of the invention. Certain amino-terminal and/orcarboxy-terminal modifications and/or peptide extensions to the coresequence can provide advantageous physical, chemical, biochemical, andpharmacological properties, such as: enhanced stability, increasedpotency and/or efficacy, resistance to serum proteases, desirablepharmacokinetic properties, and others. Peptides disclosed herein can beused therapeutically to treat disease, e.g., by altering costimulationin a patient.

Peptidomimetics (Fauchere (1986) Adv. Drug Res. 15:29; Veber andFreidinger (1985) TINS p. 392; and Evans et al. (1987) J. Med. Chem.30:1229, which are incorporated herein by reference) are usuallydeveloped with the aid of computerized molecular modeling. Peptidemimetics that are structurally similar to therapeutically usefulpeptides can be used to produce an equivalent therapeutic orprophylactic effect. Generally, peptidomimetics are structurally similarto a paradigm polypeptide (i.e., a polypeptide that has a biological orpharmacological activity), but have one or more peptide linkagesoptionally replaced by a linkage selected from the group consisting of:—CH2NH—, —CH₂S—, —CH2-CH2-, —CH═CH— (cis and trans), —COCH2-,—CH(OH)CH2-, and —CH2SO—, by methods known in the art and furtherdescribed in the following references: Spatola, A. F. in “Chemistry andBiochemistry of Amino Acids, Peptides, and Proteins” Weinstein, B., ed.,Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March1983), Vol. 1, Issue 3, “Peptide Backbone Modifications” (generalreview); Morley, J. S. (1980) Trends Pharm. Sci. pp. 463-468 (generalreview); Hudson, D. et al. (1979) Int. J. Pept. Prot. Res. 14:177-185(—CH2NH—, CH2CH2-); Spatola, A. F. et al. (1986) Life Sci. 38:1243-1249(—CH2-S); Hann, M. M. (1982) J. Chem. Soc. Perkin Trans. I. 307-314(—CH—CH—, cis and trans); Almquist, R. G. et al. (190) J. Med. Chem.23:1392-1398 (—COCH2-); Jennings-White, C. et al. (1982) TetrahedronLett. 23:2533 (—COCH2-); Szelke, M. et al. European Appln. EP 45665(1982) CA: 97:39405 (1982)(—CH(OH)CH2-); Holladay, M. W. et al. (1983)Tetrahedron Lett. (1983) 24:4401-4404 (—C(OH)CH2-); and Hruby, V. J.(1982) Life Sci. (1982) 31:189-199 (—CH2-S—); each of which isincorporated herein by reference. A particularly preferred non-peptidelinkage is —CH2NH—. Such peptide mimetics may have significantadvantages over polypeptide embodiments, including, for example: moreeconomical production, greater chemical stability, enhancedpharmacological properties (half-life, absorption, potency, efficacy,etc.), altered specificity (e.g., a broad-spectrum of biologicalactivities), reduced antigenicity, and others. Labeling ofpeptidomimetics usually involves covalent attachment of one or morelabels, directly or through a spacer (e.g., an amide group), tonon-interfering position(s) on the peptidomimetic that are predicted byquantitative structure-activity data and/or molecular modeling. Suchnon-interfering positions generally are positions that do not formdirect contacts with the macropolypeptides(s) to which thepeptidomimetic binds to produce the therapeutic effect. Derivatization(e.g., labeling) of peptidomimetics should not substantially interferewith the desired biological or pharmacological activity of thepeptidomimetic.

Also encompassed by the present invention are small molecules which canmodulate (either enhance or inhibit) interactions, e.g., betweenbiomarkers described herein or listed in Table 1 and their naturalbinding partners. The small molecules of the present invention can beobtained using any of the numerous approaches in combinatorial librarymethods known in the art, including: spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. (Lam,K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andin Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds can be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner USP '409), plasmids (Cull etal. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scottand Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.). Compounds can be screened in cell based or non-cell basedassays. Compounds can be screened in pools (e.g. multiple compounds ineach testing sample) or as individual compounds.

The invention also relates to chimeric or fusion proteins of thebiomarkers of the invention, including the biomarkers listed in Table 1,or fragments thereof. As used herein, a “chimeric protein” or “fusionprotein” comprises one or more biomarkers of the invention, includingone or more biomarkers listed in Table 1, or a fragment thereof,operatively linked to another polypeptide having an amino acid sequencecorresponding to a protein which is not substantially homologous to therespective biomarker. In a preferred embodiment, the fusion proteincomprises at least one biologically active portion of one or morebiomarkers of the invention, including one or more biomarkers listed inTable 1, or fragments thereof. Within the fusion protein, the term“operatively linked” is intended to indicate that the biomarkersequences and the non-biomarker sequences are fused in-frame to eachother in such a way as to preserve functions exhibited when expressedindependently of the fusion. The “another” sequences can be fused to theN-terminus or C-terminus of the biomarker sequences, respectively.

Such a fusion protein can be produced by recombinant expression of anucleotide sequence encoding the first peptide and a nucleotide sequenceencoding the second peptide. The second peptide may optionallycorrespond to a moiety that alters the solubility, affinity, stabilityor valency of the first peptide, for example, an immunoglobulin constantregion. In another preferred embodiment, the first peptide consists of aportion of a biologically active molecule (e.g. the extracellularportion of the polypeptide or the ligand binding portion). The secondpeptide can include an immunoglobulin constant region, for example, ahuman Cγ1 domain or Cγ4 domain (e.g., the hinge, CH2 and CH3 regions ofhuman IgCγ1, or human IgCγ4, see e.g., Capon et al. U.S. Pat. Nos.5,116,964; 5,580,756; 5,844,095 and the like, incorporated herein byreference). Such constant regions may retain regions which mediateeffector function (e.g. Fc receptor binding) or may be altered to reduceeffector function. A resulting fusion protein may have alteredsolubility, binding affinity, stability and/or valency (i.e., the numberof binding sites available per polypeptide) as compared to theindependently expressed first peptide, and may increase the efficiencyof protein purification. Fusion proteins and peptides produced byrecombinant techniques can be secreted and isolated from a mixture ofcells and medium containing the protein or peptide. Alternatively, theprotein or peptide can be retained cytoplasmically and the cellsharvested, lysed and the protein isolated. A cell culture typicallyincludes host cells, media and other byproducts. Suitable media for cellculture are well known in the art. Protein and peptides can be isolatedfrom cell culture media, host cells, or both using techniques known inthe art for purifying proteins and peptides. Techniques for transfectinghost cells and purifying proteins and peptides are known in the art.

Preferably, a fusion protein of the invention is produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, for example employingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide for appropriate termini, filling-in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992).

Particularly preferred Ig fusion proteins include the extracellulardomain portion or variable region-like domain of PD-L1, TIM-3, LAG-3, orother biomarker listed in Table 1, coupled to an immunoglobulin constantregion (e.g., the Fc region). The immunoglobulin constant region maycontain genetic modifications which reduce or eliminate effectoractivity inherent in the immunoglobulin structure. For example, DNAencoding the extracellular portion of a polypeptide of interest can bejoined to DNA encoding the hinge, CH2 and CH3 regions of human IgGγ1and/or IgGγ4 modified by site directed mutagenesis, e.g., as taught inWO 97/28267.

In another embodiment, the fusion protein contains a heterologous signalsequence at its N-terminus. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a polypeptide can be increasedthrough use of a heterologous signal sequence.

The fusion proteins of the invention can be used as immunogens toproduce antibodies in a subject. Such antibodies may be used to purifythe respective natural polypeptides from which the fusion proteins weregenerated, or in screening assays to identify polypeptides which inhibitthe interactions between one or more biomarkers polypeptide or afragment thereof and its natural binding partner(s) or a fragment(s)thereof.

Also provided herein are compositions comprising one or more nucleicacids comprising or capable of expressing at least 1, 2, 3, 4, 5, 10, 20or more small nucleic acids or antisense oligonucleotides or derivativesthereof, wherein said small nucleic acids or antisense oligonucleotidesor derivatives thereof in a cell specifically hybridize (e.g., bind)under cellular conditions, with cellular nucleic acids (e.g., smallnon-coding RNAS such as miRNAs, pre-miRNAs, pri-miRNAs, miRNA*,anti-miRNA, a miRNA binding site, a variant and/or functional variantthereof, cellular mRNAs or a fragments thereof). In one embodiment,expression of the small nucleic acids or antisense oligonucleotides orderivatives thereof in a cell can inhibit expression or biologicalactivity of cellular nucleic acids and/or proteins, e.g., by inhibitingtranscription, translation and/or small nucleic acid processing of, forexample, one or more biomarkers of the invention, including one or morebiomarkers listed in Table 1, or fragment(s) thereof. In one embodiment,the small nucleic acids or antisense oligonucleotides or derivativesthereof are small RNAs (e.g., microRNAs) or complements of small RNAs.In another embodiment, the small nucleic acids or antisenseoligonucleotides or derivatives thereof can be single or double strandedand are at least six nucleotides in length and are less than about 1000,900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 24, 23, 22,21,20, 19, 18, 17, 16, 15, or 10 nucleotides in length. In anotherembodiment, a composition may comprise a library of nucleic acidscomprising or capable of expressing small nucleic acids or antisenseoligonucleotides or derivatives thereof, or pools of said small nucleicacids or antisense oligonucleotides or derivatives thereof. A pool ofnucleic acids may comprise about 2-5, 5-10, 10-20, 10-30 or more nucleicacids comprising or capable of expressing small nucleic acids orantisense oligonucleotides or derivatives thereof.

In one embodiment, binding may be by conventional base paircomplementarity, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix. In general, “antisense” refers to the range of techniquesgenerally employed in the art, and includes any process that relies onspecific binding to oligonucleotide sequences.

It is well known in the art that modifications can be made to thesequence of a miRNA or a pre-miRNA without disrupting miRNA activity. Asused herein, the term “functional variant” of a miRNA sequence refers toan oligonucleotide sequence that varies from the natural miRNA sequence,but retains one or more functional characteristics of the miRNA (e.g.cancer cell proliferation inhibition, induction of cancer cellapoptosis, enhancement of cancer cell susceptibility to chemotherapeuticagents, specific miRNA target inhibition). In some embodiments, afunctional variant of a miRNA sequence retains all of the functionalcharacteristics of the miRNA. In certain embodiments, a functionalvariant of a miRNA has a nucleobase sequence that is a least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the miRNA or precursor thereof over a region of about5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 ormore nucleobases, or that the functional variant hybridizes to thecomplement of the miRNA or precursor thereof under stringenthybridization conditions. Accordingly, in certain embodiments thenucleobase sequence of a functional variant is capable of hybridizing toone or more target sequences of the miRNA.

miRNAs and their corresponding stem-loop sequences described herein maybe found in miRBase, an online searchable database of miRNA sequencesand annotation, found on the world wide web at microrna.sanger.ac.uk.Entries in the miRBase Sequence database represent a predicted hairpinportion of a miRNA transcript (the stem-loop), with information on thelocation and sequence of the mature miRNA sequence. The miRNA stem-loopsequences in the database are not strictly precursor miRNAs(pre-miRNAs), and may in some instances include the pre-miRNA and someflanking sequence from the presumed primary transcript. The miRNAnucleobase sequences described herein encompass any version of themiRNA, including the sequences described in Release 10.0 of the miRBasesequence database and sequences described in any earlier Release of themiRBase sequence database. A sequence database release may result in there-naming of certain miRNAs. A sequence database release may result in avariation of a mature miRNA sequence.

In some embodiments, miRNA sequences of the invention may be associatedwith a second RNA sequence that may be located on the same RNA moleculeor on a separate RNA molecule as the miRNA sequence. In such cases, themiRNA sequence may be referred to as the active strand, while the secondRNA sequence, which is at least partially complementary to the miRNAsequence, may be referred to as the complementary strand. The active andcomplementary strands are hybridized to create a double-stranded RNAthat is similar to a naturally occurring miRNA precursor. The activityof a miRNA may be optimized by maximizing uptake of the active strandand minimizing uptake of the complementary strand by the miRNA proteincomplex that regulates gene translation. This can be done throughmodification and/or design of the complementary strand.

In some embodiments, the complementary strand is modified so that achemical group other than a phosphate or hydroxyl at its 5′ terminus.The presence of the 5′ modification apparently eliminates uptake of thecomplementary strand and subsequently favors uptake of the active strandby the miRNA protein complex. The 5′ modification can be any of avariety of molecules known in the art, including NH₂, NHCOCH₃, andbiotin.

In another embodiment, the uptake of the complementary strand by themiRNA pathway is reduced by incorporating nucleotides with sugarmodifications in the first 2-6 nucleotides of the complementary strand.It should be noted that such sugar modifications can be combined withthe 5′ terminal modifications described above to further enhance miRNAactivities.

In some embodiments, the complementary strand is designed so thatnucleotides in the 3′ end of the complementary strand are notcomplementary to the active strand. This results in double-strand hybridRNAs that are stable at the 3′ end of the active strand but relativelyunstable at the 5′ end of the active strand. This difference instability enhances the uptake of the active strand by the miRNA pathway,while reducing uptake of the complementary strand, thereby enhancingmiRNA activity.

Small nucleic acid and/or antisense constructs of the methods andcompositions presented herein can be delivered, for example, as anexpression plasmid which, when transcribed in the cell, produces RNAwhich is complementary to at least a unique portion of cellular nucleicacids (e.g., small RNAs, mRNA, and/or genomic DNA). Alternatively, thesmall nucleic acid molecules can produce RNA which encodes mRNA, miRNA,pre-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding site, or avariant thereof. For example, selection of plasmids suitable forexpressing the miRNAs, methods for inserting nucleic acid sequences intothe plasmid, and methods of delivering the recombinant plasmid to thecells of interest are within the skill in the art. See, for example,Zeng et al. (2002) Mol. Cell 9:1327-1333; Tuschl (2002), Nat.Biotechnol. 20:446-448; Brummelkamp et al. (2002) Science 296:550-553;Miyagishi et al. (2002) Nat. Biotechnol. 20:497-500; Paddison et al.(2002) Genes Dev. 16:948-958; Lee et al. (2002) Nat. Biotechnol.20:500-505; and Paul et al. (2002) Nat. Biotechnol. 20:505-508, theentire disclosures of which are herein incorporated by reference.

Alternatively, small nucleic acids and/or antisense constructs areoligonucleotide probes that are generated ex vivo and which, whenintroduced into the cell, results in hybridization with cellular nucleicacids. Such oligonucleotide probes are preferably modifiedoligonucleotides that are resistant to endogenous nucleases, e.g.,exonucleases and/or endonucleases, and are therefore stable in vivo.Exemplary nucleic acid molecules for use as small nucleic acids and/orantisense oligonucleotides are phosphoramidate, phosphothioate andmethylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996;5,264,564; and 5,256,775). Additionally, general approaches toconstructing oligomers useful in antisense therapy have been reviewed,for example, by Van der Krol et al. (1988) BioTechniques 6:958-976; andStein et al. (1988) Cancer Res 48:2659-2668.

Antisense approaches may involve the design of oligonucleotides (eitherDNA or RNA) that are complementary to cellular nucleic acids (e.g.,complementary to biomarkers listed in Table 1). Absolute complementarityis not required. In the case of double-stranded antisense nucleic acids,a single strand of the duplex DNA may thus be tested, or triplexformation may be assayed. The ability to hybridize will depend on boththe degree of complementarity and the length of the antisense nucleicacid. Generally, the longer the hybridizing nucleic acid, the more basemismatches with a nucleic acid (e.g., RNA) it may contain and still forma stable duplex (or triplex, as the case may be). One skilled in the artcan ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the mRNA, e.g.,the 5′ untranslated sequence up to and including the AUG initiationcodon, should work most efficiently at inhibiting translation. However,sequences complementary to the 3′ untranslated sequences of mRNAs haverecently been shown to be effective at inhibiting translation of mRNAsas well (Wagner (1994) Nature 372:333). Therefore, oligonucleotidescomplementary to either the 5′ or 3′ untranslated, non-coding regions ofgenes could be used in an antisense approach to inhibit translation ofendogenous mRNAs. Oligonucleotides complementary to the 5′ untranslatedregion of the mRNA may include the complement of the AUG start codon.Antisense oligonucleotides complementary to mRNA coding regions are lessefficient inhibitors of translation but could also be used in accordancewith the methods and compositions presented herein. Whether designed tohybridize to the 5′, 3′ or coding region of cellular mRNAs, smallnucleic acids and/or antisense nucleic acids should be at least sixnucleotides in length, and can be less than about 1000, 900, 800, 700,600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 24, 23, 22, 21,20, 19, 18,17, 16, 15, or 10 nucleotides in length.

Regardless of the choice of target sequence, it is preferred that invitro studies are first performed to quantitate the ability of theantisense oligonucleotide to inhibit gene expression. In one embodimentthese studies utilize controls that distinguish between antisense geneinhibition and nonspecific biological effects of oligonucleotides. Inanother embodiment these studies compare levels of the target nucleicacid or protein with that of an internal control nucleic acid orprotein. Additionally, it is envisioned that results obtained using theantisense oligonucleotide are compared with those obtained using acontrol oligonucleotide. It is preferred that the controloligonucleotide is of approximately the same length as the testoligonucleotide and that the nucleotide sequence of the oligonucleotidediffers from the antisense sequence no more than is necessary to preventspecific hybridization to the target sequence.

Small nucleic acids and/or antisense oligonucleotides can be DNA or RNAor chimeric mixtures or derivatives or modified versions thereof,single-stranded or double-stranded. Small nucleic acids and/or antisenseoligonucleotides can be modified at the base moiety, sugar moiety, orphosphate backbone, for example, to improve stability of the molecule,hybridization, etc., and may include other appended groups such aspeptides (e.g., for targeting host cell receptors), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al.(1987) Proc. Natl. Acad. Sci. U.S.A. 84:648-652; PCT Publication No.WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No.WO89/10134), hybridization-triggered cleavage agents. (See, e.g., Krolet al. (1988) BioTech. 6:958-976) or intercalating agents. (See, e.g.,Zon (1988) Pharm. Res. 5:539-549). To this end, small nucleic acidsand/or antisense oligonucleotides may be conjugated to another molecule,e.g., a peptide, hybridization triggered cross-linking agent, transportagent, hybridization-triggered cleavage agent, etc.

Small nucleic acids and/or antisense oligonucleotides may comprise atleast one modified base moiety which is selected from the groupincluding but not limited to 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxytiethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Small nucleic acids and/or antisenseoligonucleotides may also comprise at least one modified sugar moietyselected from the group including but not limited to arabinose,2-fluoroarabinose, xylulose, and hexose.

In certain embodiments, a compound comprises an oligonucleotide (e.g., amiRNA or miRNA encoding oligonucleotide) conjugated to one or moremoieties which enhance the activity, cellular distribution or cellularuptake of the resulting oligonucleotide. In certain such embodiments,the moiety is a cholesterol moiety (e.g., antagomirs) or a lipid moietyor liposome conjugate. Additional moieties for conjugation includecarbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine,anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.In certain embodiments, a conjugate group is attached directly to theoligonucleotide. In certain embodiments, a conjugate group is attachedto the oligonucleotide by a linking moiety selected from amino,hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triplebonds), 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), 6-aminohexanoicacid (AHEX or AHA), substituted C1-C10 alkyl, substituted orunsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10alkynyl. In certain such embodiments, a substituent group is selectedfrom hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In certain such embodiments, the compound comprises the oligonucleotidehaving one or more stabilizing groups that are attached to one or bothtermini of the oligonucleotide to enhance properties such as, forexample, nuclease stability. Included in stabilizing groups are capstructures. These terminal modifications protect the oligonucleotidefrom exonuclease degradation, and can help in delivery and/orlocalization within a cell. The cap can be present at the 5′-terminus(5′-cap), or at the 3′-terminus (3′-cap), or can be present on bothtermini. Cap structures include, for example, inverted deoxy abasiccaps.

Suitable cap structures include a 4′,5′-methylene nucleotide, a1-(beta-D-erythrofuranosyl) nucleotide, a 4′-thio nucleotide, acarbocyclic nucleotide, a 1,5-anhydrohexitol nucleotide, anL-nucleotide, an alpha-nucleotide, a modified base nucleotide, aphosphorodithioate linkage, a threo-pentofuranosyl nucleotide, anacyclic 3′,4′-seco nucleotide, an acyclic 3,4-dihydroxybutyl nucleotide,an acyclic 3,5-dihydroxypentyl nucleotide, a 3′-3′-inverted nucleotidemoiety, a 3′-3′-inverted abasic moiety, a 3′-2′-inverted nucleotidemoiety, a 3′-2′-inverted abasic moiety, a 1,4-butanediol phosphate, a3′-phosphoramidate, a hexylphosphate, an aminohexyl phosphate, a3′-phosphate, a 3′-phosphorothioate, a phosphorodithioate, a bridgingmethylphosphonate moiety, and a non-bridging methylphosphonate moiety5′-amino-alkyl phosphate, a 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate, a 6-aminohexyl phosphate, a 1,2-aminododecylphosphate, a hydroxypropyl phosphate, a 5′-5′-inverted nucleotidemoiety, a 5′-5′-inverted abasic moiety, a 5′-phosphoramidate, a5′-phosphorothioate, a 5′-amino, a bridging and/or non-bridging5′-phosphoramidate, a phosphorothioate, and a 5′-mercapto moiety.

Small nucleic acids and/or antisense oligonucleotides can also contain aneutral peptide-like backbone. Such molecules are termed peptide nucleicacid (PNA)-oligomers and are described, e.g., in Perry-O'Keefe et al.(1996) Proc. Natl. Acad. Sci. U.S.A. 93:14670 and in Eglom et al. (1993)Nature 365:566. One advantage of PNA oligomers is their capability tobind to complementary DNA essentially independently from the ionicstrength of the medium due to the neutral backbone of the DNA. In yetanother embodiment, small nucleic acids and/or antisenseoligonucleotides comprises at least one modified phosphate backboneselected from the group consisting of a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

In a further embodiment, small nucleic acids and/or antisenseoligonucleotides are a-anomeric oligonucleotides. An α-anomericoligonucleotide forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual b-units, the strandsrun parallel to each other (Gautier et al. (1987) Nucl. Acids Res.15:6625-6641). The oligonucleotide is a 2′-0-methylribonucleotide (Inoueet al. (1987) Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNAanalogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

Small nucleic acids and/or antisense oligonucleotides of the methods andcompositions presented herein may be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988) Nucl. Acids Res. 16:3209,methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al. (1988) Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc. For example, an isolated miRNA can bechemically synthesized or recombinantly produced using methods known inthe art. In some instances, miRNA are chemically synthesized usingappropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNAmolecules or synthesis reagents include, e.g., Proligo (Hamburg,Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical(part of Perbio Science, Rockford, Ill., USA), Glen Research (Sterling,Va., USA), ChemGenes (Ashland, Mass., USA), Cruachem (Glasgow, UK), andExiqon (Vedbaek, Denmark).

Small nucleic acids and/or antisense oligonucleotides can be deliveredto cells in vivo. A number of methods have been developed for deliveringsmall nucleic acids and/or antisense oligonucleotides DNA or RNA tocells; e.g., antisense molecules can be injected directly into thetissue site, or modified antisense molecules, designed to target thedesired cells (e.g., antisense linked to peptides or antibodies thatspecifically bind receptors or antigens expressed on the target cellsurface) can be administered systematically.

In one embodiment, small nucleic acids and/or antisense oligonucleotidesmay comprise or be generated from double stranded small interfering RNAs(siRNAs), in which sequences fully complementary to cellular nucleicacids (e.g. mRNAs) sequences mediate degradation or in which sequencesincompletely complementary to cellular nucleic acids (e.g., mRNAs)mediate translational repression when expressed within cells. In anotherembodiment, double stranded siRNAs can be processed into single strandedantisense RNAs that bind single stranded cellular RNAs (e.g., microRNAs)and inhibit their expression. RNA interference (RNAi) is the process ofsequence-specific, post-transcriptional gene silencing in animals andplants, initiated by double-stranded RNA (dsRNA) that is homologous insequence to the silenced gene. in vivo, long dsRNA is cleaved byribonuclease III to generate 21- and 22-nucleotide siRNAs. It has beenshown that 21-nucleotide siRNA duplexes specifically suppress expressionof endogenous and heterologous genes in different mammalian cell lines,including human embryonic kidney (293) and HeLa cells (Elbashir et al.(2001) Nature 411:494-498). Accordingly, translation of a gene in a cellcan be inhibited by contacting the cell with short double stranded RNAshaving a length of about 15 to 30 nucleotides or of about 18 to 21nucleotides or of about 19 to 21 nucleotides. Alternatively, a vectorencoding for such siRNAs or short hairpin RNAs (shRNAs) that aremetabolized into siRNAs can be introduced into a target cell (see, e.g.,McManus et al. (2002) RNA 8:842; Xia et al. (2002) Nature Biotechnology20:1006; and Brummelkamp et al. (2002) Science 296:550). Vectors thatcan be used are commercially available, e.g., from OligoEngine under thename pSuper RNAi System™.

Ribozyme molecules designed to catalytically cleave cellular mRNAtranscripts can also be used to prevent translation of cellular mRNAsand expression of cellular polypeptides, or both (See, e.g., PCTInternational Publication WO90/11364, published Oct. 4, 1990; Sarver etal. (1990) Science 247:1222-1225 and U.S. Pat. No. 5,093,246). Whileribozymes that cleave mRNA at site specific recognition sequences can beused to destroy cellular mRNAs, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach (1988) Nature 334:585-591. The ribozymemay be engineered so that the cleavage recognition site is located nearthe 5′ end of cellular mRNAs; i.e., to increase efficiency and minimizethe intracellular accumulation of non-functional mRNA transcripts.

The ribozymes of the methods presented herein also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug et al. (1984) Science 224:574-578; Zaug et al.(1986) Science 231:470-475; Zaug et al. (1986) Nature 324:429-433; WO88/04300; and Been et al. (1986) Cell 47:207-216). The Cech-typeribozymes have an eight base pair active site which hybridizes to atarget RNA sequence whereafter cleavage of the target RNA takes place.The methods and compositions presented herein encompasses thoseCech-type ribozymes which target eight base-pair active site sequencesthat are present in cellular genes.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.). Apreferred method of delivery involves using a DNA construct “encoding”the ribozyme under the control of a strong constitutive pol III or polII promoter, so that transfected cells will produce sufficientquantities of the ribozyme to destroy endogenous cellular messages andinhibit translation. Because ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

Nucleic acid molecules to be used in triple helix formation for theinhibition of transcription of cellular genes are preferably singlestranded and composed of deoxyribonucleotides. The base composition ofthese oligonucleotides should promote triple helix formation viaHoogsteen base pairing rules, which generally require sizable stretchesof either purines or pyrimidines to be present on one strand of aduplex. Nucleotide sequences may be pyrimidine-based, which will resultin TAT and CGC triplets across the three associated strands of theresulting triple helix. The pyrimidine-rich molecules provide basecomplementarity to a purine-rich region of a single strand of the duplexin a parallel orientation to that strand. In addition, nucleic acidmolecules may be chosen that are purine-rich, for example, containing astretch of G residues. These molecules will form a triple helix with aDNA duplex that is rich in GC pairs, in which the majority of the purineresidues are located on a single strand of the targeted duplex,resulting in CGC triplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′, 3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

Small nucleic acids (e.g., miRNAs, pre-miRNAs, pri-miRNAs, miRNA*,anti-miRNA, or a miRNA binding site, or a variant thereof), antisenseoligonucleotides, ribozymes, and triple helix molecules of the methodsand compositions presented herein may be prepared by any method known inthe art for the synthesis of DNA and RNA molecules. These includetechniques for chemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculesmay be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Moreover, various well-known modifications to nucleic acid molecules maybe introduced as a means of increasing intracellular stability andhalf-life. Possible modifications include but are not limited to theaddition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule or the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages within the oligodeoxyribonucleotide backbone. One of skill inthe art will readily understand that polypeptides, small nucleic acids,and antisense oligonucleotides can be further linked to another peptideor polypeptide (e.g., a heterologous peptide), e.g., that serves as ameans of protein detection. Non-limiting examples of label peptide orpolypeptide moieties useful for detection in the invention include,without limitation, suitable enzymes such as horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;epitope tags, such as FLAG, MYC, HA, or HIS tags; fluorophores such asgreen fluorescent protein; dyes; radioisotopes; digoxygenin; biotin;antibodies; polymers; as well as others known in the art, for example,in Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz (Editor),Plenum Pub Corp, 2nd edition (July 1999).

The modulatory agents described herein (e.g., antibodies, smallmolecules, peptides, fusion proteins, or small nucleic acids) can beincorporated into pharmaceutical compositions and administered to asubject in vivo. The compositions may contain a single such molecule oragent or any combination of agents described herein. “Single activeagents” described herein can be combined with other pharmacologicallyactive compounds (“second active agents”) known in the art according tothe methods and compositions provided herein. It is believed thatcertain combinations work synergistically in the treatment of conditionsthat would benefit from the modulation of immune responses. Secondactive agents can be large molecules (e.g., proteins) or small molecules(e.g., synthetic inorganic, organometallic, or organic molecules). Forexample, anti-PD-L1 and anti-TIM-3 antibodies can be further combinedwith anti-LAG-3, anti-PD-1, anti-PD-L2, anti-CTLA4, etc. antibodies orcombinations thereof.

Examples of large molecule active agents include, but are not limitedto, hematopoietic growth factors, cytokines, and monoclonal andpolyclonal antibodies. Typical large molecule active agents arebiological molecules, such as naturally occurring or artificially madeproteins. Proteins that are particularly useful in this inventioninclude proteins that stimulate the survival and/or proliferation ofhematopoietic precursor cells and immunologically active poietic cellsin vitro or in vivo. Others stimulate the division and differentiationof committed erythroid progenitors in cells in vitro or in vivo.Particular proteins include, but are not limited to: interleukins, suchas IL-2 (including recombinant IL-2 (“rIL2”) and canarypox IL-2), IL-10,IL-12, and IL-18; interferons, such as interferon alfa-2a, interferonalfa-2b, interferon alpha-n1, interferon alpha-n3, interferon beta-Ia,and interferon gamma-Ib; GM-CF and GM-CSF; and EPO.

Particular proteins that can be used in the methods and compositionsprovided herein include, but are not limited to: filgrastim, which issold in the United States under the trade name Neupogen® (Amgen,Thousand Oaks, Calif.); sargramostim, which is sold in the United Statesunder the trade name Leukine® (Immunex, Seattle, Wash.); and recombinantEPO, which is sold in the United States under the trade name Epogen®(Amgen, Thousand Oaks, Calif.). Recombinant and mutated forms of GM-CSFcan be prepared as described in U.S. Pat. Nos. 5,391,485; 5,393,870; and5,229,496; all of which are incorporated herein by reference.Recombinant and mutated forms of G-CSF can be prepared as described inU.S. Pat. Nos. 4,810,643; 4,999,291; 5,528,823; and 5,580,755; all ofwhich are incorporated herein by reference.

Similarly, chemotherapeutic agents are well known in the art. Forexample, chemotherapeutic agents include alkylating agents such asthiotepa and cyclophosphamide (Cytoxan™); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; emylerumines and memylamelaminesincluding alfretamine, triemylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, and trimemylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (includingsynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (articularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, foremustine, lomustine, nimustine,ranimustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinphili); dynemicin, including dynemicin A; bisphosphonates, such asclodronate; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromomophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carrninomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (Adramycin™) (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as demopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogues such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replinisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; hestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformthine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK™; razoxane;rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-tricUorotriemylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethane; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiopeta; taxoids,e.g., paclitaxel (Taxol™, Bristol Meyers Squibb Oncology, Princeton,N.J.) and docetaxel (Taxoteret™, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine (Gemzar™); 6-thioguanine; mercaptopurine;methotrexate; platinum analogs such as cisplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitroxantrone;vancristine; vinorelbine (Navelbine™); novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeoloda; ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above. Alsoincluded in the definition of “chemotherapeutic agent” are anti-hormonalagents that act to regulate or inhibit hormone action on tumors such asanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including Nolvadex™), raloxifene,droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,onapristone, and toremifene (Fareston™); inhibitors of the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrolacetate (Megace™), exemestane, formestane, fadrozole, vorozole(Rivisor™), letrozole (Femara™), and anastrozole (Arimidex™); andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprohde,and goserelin; and pharmaceutically acceptable salts, acids orderivatives of any of the above. In some embodiments, the inhibitordownregulates Rac 1 output. Additional examples of chemotherapeutic andother anti-cancer agents are described in US Pat. Publs. 2013/0239239and 2009/0053224.

b. Pharmaceutical Compositions

Agents that modulate (e.g., inhibit or block) the function of PD-1 orPD-L1 and LAG-3, CTLA-4 or TIM-3, including, e.g., blocking antibodies,peptides, fusion proteins, or small molecules, can be incorporated intopharmaceutical compositions suitable for administration to a subject.Such compositions typically comprise the antibody, peptide, fusionprotein or small molecule and a pharmaceutically acceptable carrier. Asused herein, “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions. A pharmaceutical composition of the invention isformulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g.,intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerin, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition should be sterile and should be fluid to theextent that easy syringeability exists. It must be stable under theconditions of manufacture and storage and should be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, modulatory agents are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations should be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by, and directlydependent on, the unique characteristics of the active compound, theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

The above described modulating agents may be administered it he form ofexpressible nucleic acids which encode said agents. Such nucleic acidsand compositions in which they are contained, are also encompassed bythe present invention. For instance, the nucleic acid molecules of theinvention can be inserted into vectors and used as gene therapy vectors.Gene therapy vectors can be delivered to a subject by, for example,intravenous injection, local administration (see U.S. Pat. No.5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994)Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

c. Prophylactic Methods

In one aspect, the present invention provides a method for preventing ina subject, a cancer, such as a hematologic cancer like multiple myeloma,associated with a less than desirable immune response. Subjects at riskfor such a disease can be identified, for example, by any or acombination of diagnostic or prognostic assays known in the art.Administration of a prophylactic agent(s) can occur prior to themanifestation of symptoms associated with an unwanted or less thandesirable immune response. The appropriate agent(s) used for treatment(e.g. antibodies, peptides, fusion proteins or small molecules) can bedetermined based on clinical indications and can be identified usingdiagnostic assays well known in the art, as well as those describedherein.

d. Therapeutic Methods

Another aspect of the invention pertains to therapeutic methods ofmodulating an immune response, e.g., by inhibiting or blocking thefunction of PD-1 or PD-L1 and LAG-3, CTLA-4 or TIM-3.

Modulatory methods of the present invention involve contacting a cellwith an agent that inhibits or blocks the function of PD-1 or PD-L1 andLAG-3, CTLA-4 or TIM-3. Exemplary agents useful in such methods aredescribed above. Such agents can be administered in vitro or ex vivo(e.g., by contacting the cell with the agent) or, alternatively, in vivo(e.g., by administering the agent to a subject). As such, the presentinvention provides methods useful for treating an individual afflictedwith a cancer, such as a hematologic cancer like multiple myeloma.

Agents that upregulate immune responses can be in the form of enhancingan existing immune response or eliciting an initial immune response.Thus, enhancing an immune response using the subject compositions andmethods is useful for treating cancer, but can also be useful fortreating an infectious disease (e.g., bacteria, viruses, or parasites),a parasitic infection, and an immunosuppressive disease.

Immune responses can also be enhanced in an infected patient through anex vivo approach, for instance, by removing immune cells from thepatient, contacting immune cells in vitro with an agent described hereinand reintroducing the in vitro stimulated immune cells into the patient.

In certain instances, it may be desirable to further administer otheragents that upregulate immune responses, for example, forms of other B7family members that transduce signals via costimulatory receptors, inorder to further augment the immune response.

Agents that upregulate an immune response can be used prophylacticallyin vaccines against various polypeptides (e.g., polypeptides derivedfrom pathogens). Immunity against a pathogen (e.g., a virus) can beinduced by vaccinating with a viral protein along with an agent thatupregulates an immune response, in an appropriate adjuvant.

In another embodiment, upregulation or enhancement of an immune responsefunction, as described herein, is useful in the induction of tumorimmunity.

In another embodiment, the immune response can be stimulated by themethods described herein, such that preexisting tolerance, clonaldeletion, and/or exhaustion (e.g., T cell exhaustion) is overcome. Forexample, immune responses against antigens to which a subject cannotmount a significant immune response, e.g., to an autologous antigen,such as a tumor specific antigens can be induced by administeringappropriate agents described herein that upregulate the immune response.In one embodiment, an autologous antigen, such as a tumor-specificantigen, can be coadministered. In another embodiment, the subjectagents can be used as adjuvants to boost responses to foreign antigensin the process of active immunization.

In one embodiment, immune cells are obtained from a subject and culturedex vivo in the presence of an agent as described herein, to expand thepopulation of immune cells and/or to enhance immune cell activation. Ina further embodiment the immune cells are then administered to asubject. Immune cells can be stimulated in vitro by, for example,providing to the immune cells a primary activation signal and acostimulatory signal, as is known in the art. Various agents can also beused to costimulate proliferation of immune cells. In one embodimentimmune cells are cultured ex vivo according to the method described inPCT Application No. WO 94/29436. The costimulatory polypeptide can besoluble, attached to a cell membrane, or attached to a solid surface,such as a bead.

In still another embodiment, agents described herein useful forupregulating immune responses can further be linked, or operativelyattached, to toxins using techniques that are known in the art, e.g.,crosslinking or via recombinant DNA techniques. Such agents can resultin cellular destruction of desired cells. In one embodiment, a toxin canbe conjugated to an antibody, such as a bispecific antibody. Suchantibodies are useful for targeting a specific cell population, e.g.,using a marker found only on a certain type of cell. The preparation ofimmunotoxins is, in general, well known in the art (see, e.g., U.S. Pat.No. 4,340,535, and EP 44167). Numerous types of disulfide-bondcontaining linkers are known which can successfully be employed toconjugate the toxin moiety with a polypeptide. In one embodiment,linkers that contain a disulfide bond that is sterically “hindered” arepreferred, due to their greater stability in vivo, thus preventingrelease of the toxin moiety prior to binding at the site of action. Awide variety of toxins are known that may be conjugated to polypeptidesor antibodies of the invention. Examples include: numerous usefulplant-, fungus- or even bacteria-derived toxins, which, by way ofexample, include various A chain toxins, particularly ricin A chain,ribosome inactivating proteins such as saporin or gelonin, a-sarcin,aspergillin, restrictocin, ribonucleases, such as placentalribonuclease, angiogenic, diphtheria toxin, and Pseudomonas exotoxin,etc. A preferred toxin moiety for use in connection with the inventionis toxin A chain which has been treated to modify or remove carbohydrateresidues, deglycosylated A chain. (U.S. Pat. No. 5,776,427). Infusion ofone or a combination of such cytotoxic agents, (e.g., ricin fusions)into a patient may result in the death of immune cells.

In yet another embodiment, the efficacy of the treatment methodsdescribed herein can be enhanced by incorporating a step oflymphodepletion prior to, concurrently with, or after the administrationof agents the inhibit or block PD-1, PD-L1, CTLA-4, TIM-3, and/or LAG-3function. For example, therapeutic benefits of administering thedescribed anti-cancer agents can be synergistically enhanced byperforming such administration after or in conjunction withlymphodepletion. Methods for achieving lymphodepletion in various formsand at various levels are well known in the art (see, for example, U.S.Pat. No. 7,138,144). For example, the term “transient lymphodepletion”refers to destruction of lymphocytes and T cells, usually prior toimmunotherapy. This can be accomplished in a number of ways, including“sublethal irradiation,” which refers to administration of one or moredoses of radiation that is generally non-lethal to all members of apopulation of subjects to which the administration is applied. Transientlymphodepletion is generally not myeloablative, as would be the case incomplete lymphodepletion, such that the subjects hematopoietic orimmunological capacity remains sufficiently intact to regenerate thedestroyed lymphocyte and T cell populations. By contrast, “lethalirradiation” occurs when the administration is generally lethal to somebut not all members of the population of subjects and “supralethalirradiation” occurs when the administion is generally lethal to allmembers of the population of subjects.

Depending on the application and purpose, transient lymphodepletion orcomplete lymphodepletion may be effected, for example, by anycombination of irradiation, treatment with a myeloablative agent, and/ortreatment with an immunosuppressive agent, according to standardprotocols. For example, biological methods include, for example,administration of immunity-suppressing cells or by administration ofbiological molecules capable of inhibiting immunoreactivity, such as,for example, Fas-ligand and CTLA4-Ig. Examples of myeloablative agentsinclude busulfan, dimethyl mileran, melphalan and thiotepa. Examples ofimmunosuppressive agents include prednisone, methyl prednisolone,azathioprine, cyclosporine A, cyclophosphamide, fludarabin, CTLA4-Ig,anti-T cell antibodies, etc.

Regarding irradiation, a sublethal dose of irradiation is generallywithin the range of 1 to 7.5 Gy whole body irradiation, a lethal dose isgenerally within the range of 7.5 to 9.5 Gy whole body irradiation, anda supralethal dose is within the range of 9.5 to 16.5 Gy whole bodyirradiation.

Depending on the purpose and application, the dose of irradiation may beadministered as a single dose or as a fractionated dose. Similarly,administering one or more doses of irradiation can be accomplishedessentially exclusively to the body part or to a portion thereof, so asto induce myeloreduction or myeloablation essentially exclusively in thebody part or the portion thereof. As is widely recognized in the art, asubject can tolerate as sublethal conditioning ultra-high levels ofselective irradiation to a body part such as a limb, which levelsconstituting lethal or supralethal conditioning when used for whole bodyirradiation (see, for example, Breitz (2002) Cancer Biother Radiopharm.17:119; Limit (1997) J. Nucl. Med. 38:1374; and Dritschilo and Sherman(1981) Environ. Health Perspect. 39:59). Such selective irradiation ofthe body part, or portion thereof, can be advantageously used to targetparticular blood compartments, such as specific lymph nodes, in treatinghematopoietic cancers.

e. Administration of Agents

The immune modulating agents of the invention are administered tosubjects in a biologically compatible form suitable for pharmaceuticaladministration in vivo, to enhance immune cell mediated immuneresponses. By “biologically compatible form suitable for administrationin vivo” is meant a form to be administered in which any toxic effectsare outweighed by the therapeutic effects. The term “subject” isintended to include living organisms in which an immune response can beelicited, e.g., mammals. Examples of subjects include humans, dogs,cats, mice, rats, and transgenic species thereof. Administration of anagent as described herein can be in any pharmacological form including atherapeutically active amount of an agent alone or in combination with apharmaceutically acceptable carrier.

Administration of a therapeutically active amount of the therapeuticcomposition of the present invention is defined as an amount effective,at dosages and for periods of time necessary, to achieve the desiredresult. For example, a therapeutically active amount of an agent mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of peptide to elicit a desiredresponse in the individual. Dosage regimens can be adjusted to providethe optimum therapeutic response. For example, several divided doses canbe administered daily or the dose can be proportionally reduced asindicated by the exigencies of the therapeutic situation.

Inhibiting or blocking the function of PD-1 or PD-L1 and LAG-3, CTLA-4or TIM-3, or in some embodiments, inhibiting or blocking a combinationof these agents, can be accomplished by combination therapy with themodulatory agents described herein. Combination therapy describes atherapy in which PD-1 or PD-L1 and LAG-3, CTLA-4 or TIM-3, are inhibitedor blocked simultaneously. Simultaneous inhibition or blockade may beachieved by administration of the modulatory agents described hereinsimultaneously (e.g., in a combination dosage form or by simultaneousadministration of single agents) or by administration of single agentsaccording to a schedule that results in effective amounts of eachmodulatory agent present in the patient at the same time.

The therapeutic agents described herein can be administered in aconvenient manner such as by injection (subcutaneous, intravenous,etc.), oral administration, inhalation, transdermal application, orrectal administration. Depending on the route of administration, theactive compound can be coated in a material to protect the compound fromthe action of enzymes, acids and other natural conditions which mayinactivate the compound. For example, for administration of agents, byother than parenteral administration, it may be desirable to coat theagent with, or co-administer the agent with, a material to prevent itsinactivation.

An agent can be administered to an individual in an appropriate carrier,diluent or adjuvant, co-administered with enzyme inhibitors or in anappropriate carrier such as liposomes. Pharmaceutically acceptablediluents include saline and aqueous buffer solutions. Adjuvant is usedin its broadest sense and includes any immune stimulating compound suchas interferon. Adjuvants contemplated herein include resorcinols,non-ionic surfactants such as polyoxyethylene oleyl ether andn-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatictrypsin inhibitor, diisopropylfluorophosphate (DEEP) and trasylol.Liposomes include water-in-oil-in-water emulsions as well asconventional liposomes (Sterna et al. (1984) J. Neuroimmunol. 7:27).

The agent may also be administered parenterally or intraperitoneally.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof, and in oils. Under ordinary conditions ofstorage and use, these preparations may contain a preservative toprevent the growth of microorganisms.

Pharmaceutical compositions of agents suitable for injectable useinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion. In all cases the composition willpreferably be sterile and must be fluid to the extent that easysyringeability exists. It will preferably be stable under the conditionsof manufacture and storage and preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it is preferable to includeisotonic agents, for example, sugars, polyalcohols such as manitol,sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating an agentof the invention (e.g., an antibody, peptide, fusion protein or smallmolecule) in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the agent plusany additional desired ingredient from a previously sterile-filteredsolution thereof.

When the agent is suitably protected, as described above, the proteincan be orally administered, for example, with an inert diluent or anassimilable edible carrier. As used herein “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the therapeutic compositions iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.“Dosage unit form”, as used herein, refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by, and directly dependent on, (a)the unique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

In one embodiment, an agent of the invention is an antibody. As definedherein, a therapeutically effective amount of antibody (i.e., aneffective dosage) ranges from about 0.001 to 30 mg/kg body weight,preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. Theskilled artisan will appreciate that certain factors may influence thedosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of an antibody can include a single treatment or,preferably, can include a series of treatments. In a preferred example,a subject is treated with antibody in the range of between about 0.1 to20 mg/kg body weight, one time per week for between about 1 to 10 weeks,preferably between 2 to 8 weeks, more preferably between about 3 to 7weeks, and even more preferably for about 4, 5, or 6 weeks. It will alsobe appreciated that the effective dosage of antibody used for treatmentmay increase or decrease over the course of a particular treatment.Changes in dosage may result from the results of diagnostic assays.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures, are incorporated herein byreference.

III. Kits

The present invention also encompasses kits for treating cancers, suchas hematologic cancers like multiple myeloma, using agents that inhibitor block PD-1 or PD-L1 and LAG-3,

CTLA-4 or TIM-3function. For example, the kit can comprise an antibodyas described herein, e.g., an antibody against PD-1, PD-L1, LAG-3,CTLA-4 and/or TIM-3, packaged in a suitable container and can furthercomprise instructions for using such antibodies to treat cancers in apatient in need thereof. The kit may also contain other components, suchas administration tools like packaged in a separate container.

EXAMPLES Example 1: Combined PD-L1 and TIM-3 Blockade as Immunotherapyfor Hematologic Cancers

a. Materials and Methods

Mice

All mice were housed in the Medical College of Wisconsin BiomedicalResource Center, an AAALAC-accredited facility. C47BL6/KaLwRij mice wereused in the experiments. All animal work was reviewed and approved bythe Medical College of Wisconsin Institutional Animal Care and UseCommittee.

Tumor Cells

The 5T33 murine multiple myeloma (MM) cell line was derived from myelomathat spontaneously arose in a C57B1/KaLwRij mouse (Radl et al. (1988)Am. J. Pathol. 132:593-597; and Manning et al. (1992) Br. J. Cancer66:1088-1093). For experiments, 5T33 cells were thawed from a largefrozen stock and cultured in RPMI 1640+10% fetal bovine serum for nolonger than 2 weeks prior to inoculation of mice. Mice were inoculatedwith tumor as follows: 2×10⁶ 5 T33 cells intravenously (i.v.).5T33-bearing mice were considered moribund and euthanized when theydeveloped paraparesis or paraplegia. Occasionally, 5T33-inoculated micedeveloped tumor masses or lesions and were euthanized when the size ofthe mass or lesion exceeded 250 mm²; other symptoms of advanced 5T33included splenomegaly, hepatomegaly, or neurologic impairment.

Antibodies

The clone designations for the antibodies used are as follows:anti-PD-L1 (clone 10F.9G2; Paterson et al. (2011) J. Immunol.187:1097-1105; Maier et al. (2007) J. Immunol. 178:2714-2720), anti-PD-1(clone 332.8H3), anti-Lag-3 (clone C9B7W; available from eBioscience ascatalog number eBioC9B7W and other manufacturers), anti-Tim-3 (clone5D12; available from EMD Millipore as catalog number MABF73). Isotypecontrol antibodies included Armenian hamster IgG and rat IgG2a kappa.

Irradiation

In general, myeloma-bearing recipient mice were given total bodyirradiation as a single sublethal (500 cGy) dose seven days aftermyeloma inoculation. Radiation was administered by a Shepherd Mark ICesium Irradiator in accordance with established guidelines.

Statistics

Survival curves were compared using the log rank (Mantel Cox) test basedon n=5-6 mice per group. P-values of <0.05 were considered significant.Statistical analysis was done using Prism version 5.0a software(GraphPad Software, La Jolla, Calif.).

Other materials and methods are described in the “Results” sectionbelow.

b. Results

Multiple myeloma is characterized by the presence of transformedneoplastic plasma cells in the bone marrow and is generally consideredto be an incurable disease. Successful treatments will likely requiremulti-faceted approaches incorporating conventional drug therapies,immunotherapy and other novel treatments. It has previously beendetermined that a combination of transient lymphodepletion (sublethalwhole body irradiation) and PD-1 blockade generated anti-myeloma T cellreactivity capable of eliminating established disease (Kearl et al.(2013) J. Immunol. 190:5620-5628; Hallett et al. (2011) Biol. BloodMarrow Transplant. 17:1133-1145). Besides expression of the immunecheckpoint protein PD-1, T cells within tumor environment may develop adysfunctional phenotype accompanied by the increased expression of othercheckpoint proteins.

It was hypothesized that the anti-myeloma effect of transientlymphodepletion and PD-1 blockade would be increased by blocking otherimmune checkpoint protein interactions.

Expression of Immune Checkpoint Proteins on T Cells in Bone Marrow ofMyeloma Bearing Mice Over Time

Accordingly, an extensive phenotypic analysis (flow cytometry) of bonemarrow and splenic tissues from myeloma-bearing mice was performed totemporally examine T cells for expression of immune checkpoint proteinsand assess the tissues for presence of immune regulatory T (Treg) cells.KaLwRij mice were inoculated with 2×10⁶ 5 T33-GFP cells intravenously.Myeloma bearing mice were euthanized between days 7 and 28 afterinoculation or when moribund (day 29-40), and femoral bone marrow cellswere harvested. Tumor cell accumulation was monitored by flow cytometry(GFP+ tumor cells) (FIG. 1A), and CD4⁺ (top) and CD8⁺ (bottom) T cellswere analyzed by flow cytometry for expression of various immunecheckpoint proteins over time (FIG. 1B). Naïve non-myeloma bearing micewere analyzed as controls. Immune checkpoint protein percentages werebased on isotype controls.

As shown in FIG. 1B, PD-1, 2B4, LAG3, and TIM-3 were the most prominentimmune checkpoint proteins present on T cells in myeloma bearing mice.As shown in FIG. 1C, a relatively large percentage of PD-1⁺ T cellsco-expressed other inhibitory checkpoint proteins such as Tim-3, Lag-3and 2B4. It was also determined that Treg cells in the tumormicroenvironment also had increased expression of PD-1 and otherinhibitory receptors (such as Tim-3, Lag-3 and 2B4 shown in FIG. 2 )compared to Tregs cells from non-myeloma bearing mice, which is believedto be related with enhanced suppressive function by these cells.

Increased Expression of Immune Checkpoint Proteins on T Cells in MiceTreated with Sublethal Whole Body Irradiation and Anti-PD-L1 Antibody

Myeloma bearing KaLwRij mice were treated with 500 cGy whole bodyirradiation 7 days after tumor cell inoculation. Treatment withanti-PD-L1 antibody or control IgG (200m i.p.) was initiated 5 dayslater and specifically given 12, 14, and 19 days after tumorinoculation. Mice were euthanized at day 21, splenocytes were harvested,and the CD8 T cells were analyzed by flow cytometry for immunecheckpoint protein expression. As shown in FIGS. 3A-3B, the frequenciesof CD8⁺Tim-3⁺, CD8⁺Lag-3⁺ and CD8⁺2B4⁺ cells in spleens of anti-PD-L1antibody treated mice were higher compared with spleens of controlantibody treated.

Blocking PD-L1 in Combination with Tim-3 after Lymphodepleting WholeBody Irradiation Synergistically Improved Survival

It was then examined whether blocking various immune checkpoint proteinscould provide additive or synergistic anti-myeloma effects when combinedwith PD-L1 blockade (FIG. 4 ). In this Example, the combined blockade ofPD-1 and TIM-3 was most effective and proved to be synergistic, asmyeloma was surprisingly rejected in 100% of these mice (FIG. 5 ).Inhibition of certain other immune checkpoint proteins, either alone orin combinations, did not produce such robust therapeutic benefits (FIGS.6-8 ).

Thus, the data indicate that dual blockade of PD-L1 and TIM-3 representsa surprisingly and unexpectedly potent immunotherapeutic interventionfor treating hematologic cancers, such as multiple myeloma.

Example 2: Combined Immune Checkpoint Protein Blockade andLymphodepletion as Immunotherapy for Hematologic Cancers

a. Materials and methods are essentially the same as described inExample 1 unless specifically indicated below in the “Results” sectionbelow.

b. Results

As shown in this Example, combined immune checkpoint protein blockadeand lymphodepletion provide an effective immunotherapy for hematologiccancers such as myeloma.

Blocking of PD-L1 in Combination with Tim-3, Lag-3 or CTLA-4 afterLymphodepleting Whole Body Irradiation Synergistically Improved Survival

FIG. 9A depicts the experimental design. KaLwRij mice received 500 cGyirradiation 7 days after tumor cell inoculation. The treatment withblocking antibody or control IgG (200 μg i.p.) was initiated 5 dayslater and specifically given 12, 14, 19, 21, 26, and 28 days after tumorinoculation.

As shown in FIGS. 9B-9D, blocking PD-L1 in combination with Tim-3 (FIG.9B), Lag-3 (FIG. 9B), or CTLA-4 (FIG. 9C) after lymphodepleting wholebody irradiation synergistically improved survival of myeloma bearingmice, whereas blocking PD-L1 in combination with CD48 (FIG. 9D) did nothave synergistic effect on survival. As shown in FIG. 9E, 100% ofre-challenged mice that had received anti-PD-L1 antibody alone, or thecombination of anti-PD-L1 antibody with anti-Tim-3 antibody, anti-Lag-3antibody, or anti-CTLA-4 antibody, survived to day 110, compared tocontrol mice.

Combined Checkpoint Blockade after Lymphodepleting Whole BodyIrradiation Increased Frequencies of Tumor-Reactive T Cells

The experimental design shown in FIG. 9A was used. CD4⁺ or CD8⁺ T cellswere isolated from spleens and bone marrow 21 days after tumor cellinoculation (i.e., 14 days after irradiation) in mice treated withcontrol IgG, anti-PD-L1 antibody only, or the combination of anti-PD-L1antibody with anti-Tim-3 antibody, anti-Lag-3 antibody, or anti-CTLA-4antibody. The CD8⁺ or CD4⁺ T cells were tested in IFN-γ ELISPOT assaysusing 5T33 or MHC class II+ 5T33 tumor cells as stimulators,respectively, to determine tumor-reactive IFN-γ-secreting cellfrequencies.

As shown in FIG. 10A, the frequencies of tumor-reactive CD8⁺ and CD4⁺ Tcells were increased in the spleens (top row) and bone marrow (bottomrow) of mice treated with combinations of immune checkpoint proteinblockade. Combined checkpoint blockade, such as the combination ofanti-PD-L1 antibody with anti-Tim-3 antibody, anti-Lag-3 antibody, oranti-CTLA-4 antibody, increased frequencies of tumor-reactive T cells.

Combined Checkpoint Blockade after Lymphodepleting Whole BodyIrradiation Increased Cytokine Production by CD8⁺ T Cells

CD8⁺ T cells purified from the spleens of myeloma bearing mice treatedwith anti-PD-L1 antibody only, or the combination of anti-PD-L1 antibodywith anti-Tim-3 antibody, anti-Lag-3 antibody or anti-CTLA4 antibody,were stimulated with 5T33 for 48 hours. Supernatants were collected andcytokine levels from were determined using a multiplex cytokine assay.As shown in FIG. 10B, combined checkpoint blockade, such as thecombination of anti-PD-L1 antibody with anti-Tim-3 antibody, anti-Lag-3antibody, or anti-CTLA-4 antibody, increased the production of cytokines(e.g., IL-2, IFN-γ and GM-CSF) by CD8⁺ T cells.

Combined Blockade of Immune Checkpoint Proteins Increased PD-1Expression on CD8⁺ T Cells as Well as Increased Frequency of TumorSpecific Cytotoxic T Lymphocytes

The experimental design in FIG. 9A was used. CD8⁺ T cells were isolatedfrom spleens and bone marrow 21 days after tumor cell inoculation inmice treated with control IgG, anti-PD-L1 antibody only, or thecombination of anti-PD-L1 antibody with anti-Tim-3 antibody, anti-Lag-3antibody, or anti-CTLA4 antibody.

FIG. 11A shows increased expression of PD-1 on gated CD8⁺ T cells fromspleens and bone marrow (BM) of mice treated with different blockingantibodies or control IgG.

The CD8⁺ T cells were assayed in IFN-γ ELISPOT assays with tumor cellstimulators to determine tumor-reactive IFN-γ-secreting cell frequenciesin the presence of anti-PD-L1 antibody or control IgG (10 μg/ml). Asshown in FIG. 11B, in the presence of blocking anti-PD-L1 antibody invitro, all combinations of blocking antibodies in vivo resulted insignificantly increased cytotoxic T lymphocyte (CTL) frequencies versusblockade of PD-L1 only (p<0.001).

Combined Blockade of Immune Checkpoint Proteins Enhanced Th1 and Th2Cytokine Secretion

The experimental design shown in FIG. 9A was used. CD4⁺ T cells wereisolated from spleens 21 days after tumor cell inoculation (i.e., 14days after irradiation) in myeloma bearing mice treated with controlanti-PD-L1, or the combination of anti-PD-L1 antibody with anti-Tim-3antibody, anti-Lag-3 antibody, or anti-CTLA4 antibody. CD4⁺ T cellspurified from the spleen were stimulated with MHC class II negative5T33-WT, or MHC class II positive 5T33-CIITA, or no stimulation for 48hours. Supernatants were collected and cytokine (IFN-γ, IL-4 and IL-5)levels from were determined using a multiplex cytokine assay.

As shown in FIG. 12 , combined blockade of immune checkpoint proteinssuch as anti-PD-L1 and anti-CTLA-4 antibodies enhanced Th1 and Th2cytokine secretion.

Expression of Immune Checkpoints on T Cells in Mice with OtherHematologic Cancers

Murine hematologic cancers other than myeloma express PD-L1 and respondto whole body irradiation plus PD-L1 blockade (Kearl et al. (2013) J.Immunol. 190:5620-5628).

To examine the expression of immune checkpoint proteins on T cells inmice with other hematologic cancers, mice were injected i.v. with A20 Bcell lymphoma cells, C1498 acute myeloid leukemia cells, or EL4 lymphomacells (x-axis in FIG. 13 ). Bone marrow was collected from moribundanimals. CD4⁺ (top row in FIG. 13 ) and CD8⁺ (bottom row in FIG. 13 ) Tcells in the bone marrow were analyzed for expression of checkpointproteins PD-1, Tim-3, Lag-3 and 2B4 by flow cytometry (n=4-5 mice foreach). Naïve non-cancer-bearing mice were used as controls. FIG. 13shows expression of PD-1, Tim-3, Lag-3 and 2B4 on T cells in micebearing the indicated hematologic cancer cells. A correlation can existbetween the T cell expression profile of these checkpoint proteins andincreased anti-tumor response after co-blockade of the respectivepathway(s) in hematologic malignancy models other than myeloma.

A Model of Combined Immune Checkpoint Blockade and Lymphodepleting WholeBody Irradiation for Treating Hematologic Cancers

A working model of combined immune checkpoint blockade andlymphodepleting whole body irradiation is illustrated in FIG. 14 . Inhematologic cancers, dysfunctional antigen-activated T cells (e.g., CD4⁺and CD8⁺ T cells) are unable to kill cancer cells. Lymphopenia-induced Tcell proliferation allows for reactivation of those T cells. Forreactivated T cells to remain functional and kill cancer cells, immunecheckpoint proteins must be blocked. Lymphopenic environment can beachieved by low-dose whole body irradiation (WBI). Lymphodepletingchemotherapy or low doses of T cell-depleting antibodies can also beused instead of whole body irradiation.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

Also incorporated by reference in their entirety are any polynucleotideand polypeptide sequences which reference an accession numbercorrelating to an entry in a public database, such as those maintainedby The Institute for Genomic Research (TIGR) on the world wide web attigr.org and/or the National Center for Biotechnology Information (NCBI)on the World Wide Web at ncbi.nlm.nih.gov.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method of treating a subject afflicted with ahematologic cancer comprising administering to the subject atherapeutically effective amount of: (i) a first inhibitor that is aninhibitor of PD-L1 or PD-1 and (ii) a second inhibitor that is aninhibitor of LAG-3; and wherein the method further comprises a step oftransient lymphodepletion.
 2. The method of claim 1 wherein an inhibitorof PD-L1 is administered in combination with an inhibitor of LAG-3. 3.The method of claim 1 wherein an inhibitor of PD-1 is administered incombination with an inhibitor of LAG-3.
 4. The method of claim 1,wherein the PD-1, PD-L1, or LAG-3 inhibitor: (a) is an antibody orantigen-binding fragment thereof, that binds to one or more targetsselected from the group consisting of PD-1, PD-L1, and LAG-3; or (b) isa bispecific or multispecific antibody, or antigen binding fragmentthereof, selective for (i) PD-1 or PD-L1 and (ii) LAG-3.
 5. The methodof claim 1, wherein a combination of inhibitors comprising a firstinhibitor that selectively inhibits or blocks PD-1 or PD-L1 and a secondinhibitor that selectively inhibits or blocks LAG-3 is administered. 6.The method of claim 5, wherein the first inhibitor, second inhibitor, orboth inhibitors, is an antibody or an antigen binding fragment thereof,which specifically binds to (i) PD-1 or PD-L1 and/or (ii) LAG-3.
 7. Themethod of claim 6, wherein: (a) said antibody, or antigen bindingfragment thereof, is murine, chimeric, humanized, composite, or human;or (b) said antibody, or antigen binding fragment thereof, is detectablylabeled, comprises an effector domain, comprises an Fc domain, and/or isselected from the group consisting of Fv, Fav, F(ab′)2), Fab′, dsFv,scFv, sc(Fv)2, and diabodies fragments.
 8. The method of claim 1,wherein said first and/or second inhibitor is administered in apharmaceutically acceptable formulation.
 9. The method of claim 1,further comprising administering to the subject a therapeutic agent fortreating the hematologic cancer.
 10. The method of claim 1, wherein: (a)sublethal whole body irradiation is used for transient lymphodepletion;or (b) the step of lymphodepletion occurs before, concurrently with, orafter the step of first and/or second inhibitor administration.
 11. Themethod of claim 1, wherein the hematologic cancer is selected from thegroup consisting of: (a) multiple myeloma, acute lymphocytic leukemia,acute myeloid leukemia, chronic lymphocytic leukemia, small lymphocyticlymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, mantle celllymphoma, follicular lymphoma, Waldenstrom's macroglobulinemia, B-celllymphoma and diffuse large B-cell lymphoma, precursor B-lymphoblasticleukemia/lymphoma, B-cell chronic lymphocytic leukemia/small lymphocyticlymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma,splenic marginal zone B-cell lymphoma (with or without villouslymphocytes), hairy cell leukemia, plasma cell myeloma/plasmacytoma,extranodal marginal zone B-cell lymphoma of the MALT type, nodalmarginal zone B-cell lymphoma (with or without monocytoid B cells),Burkitt's lymphoma; precursor T-lymphoblastic lymphoma/leukemia, T-cellprolymphocytic leukemia, T-cell granular lymphocytic leukemia,aggressive NK cell leukemia, adult T-cell lymphoma/leukemia (HTLV1-positive), nasal-type extranodal NK/T-cell lymphoma, enteropathy-typeT-cell lymphoma, hepatosplenic γ-δ T-cell lymphoma, subcutaneouspanniculitis-like T-cell lymphoma, mycosis fungoides/Sezary syndrome,anaplastic large cell lymphoma (T/null cell, primary cutaneous type),anaplastic large cell lymphoma (T-/null-cell, primary systemic type),peripheral T-cell lymphoma not otherwise characterized,angioimmunoblastic T-cell lymphoma, polycythemia vera (PV),myelodysplastic syndrome (MDS), indolent Non-Hodgkin's Lymphoma (iNHL),and aggressive Non-Hodgkin's Lymphoma (aNHL); or (b) B-cell lymphoma,myeloid leukemia, and multiple myeloma.
 12. The method of claim 1,wherein the subject is a human.